FRET detection method and device

ABSTRACT

When FRET efficiency is measured quantitatively by removing uncertain elements of fluorescence detection information, calibration information prestored in a storage means while including at least the leak rate of donor fluorescence component emitted from a donor molecule, the leak rate of acceptor fluorescence component emitted from an acceptor molecule, and the non-FRET fluorescence lifetime of the donor fluorescence component when FRET is not generated out of the fluorescence of a measurement object sample is acquired. The FRET fluorescence lifetime of the donor fluorescence component is then determined using the intensity information and phase information of fluorescence of the measurement object sample, the leak rate of donor fluorescence component and the leak rate of acceptor fluorescence component, thus determining the FRET fluorescence efficiency.

TECHNICAL FIELD

The present invention relates to a method of and a device for detectingFRET (Fluorescence Resonance Energy Transfer), in which the energy of adonor molecule transfers to the energy of an acceptor molecule. Morespecifically, the present invention relates to a FRET detectiontechnology for detecting interaction between a pair of a donor molecule(fluorescent molecule) and an acceptor molecule (fluorescent molecule)using fluorescence.

BACKGROUND ART

Analysis of protein functions has recently become important aspost-genome related technology in the medical, pharmaceutical, and foodindustries. Particularly, in order to analyze actions of cells, it isnecessary to research interactions (binding and separation) between atype of protein and another type of protein (or a low molecule compound)which are living substances in a living cell.

The interactions between a type of protein and another type of protein(or a low molecule compound) have been recently analyzed using afluorescence resonance energy transfer (FRET) phenomenon. Interactionsbetween molecules are herein detected within a range of severalnanometers using fluorescence. Such interaction detection using the FRETphenomenon is mainly performed with a microscope system.

For example, Japanese Laid-Open Patent Application No. 2005-207823discloses a single molecule fluorescence analysis using FRET. JapaneseLaid-Open Patent Application No. 2005-207823 proposes a fluorescencespectral analysis method in which a fluorescence correlation analysismethod or a fluorescence-intensity distribution analysis is performedbased on received fluorescence using the FRET phenomenon.

DISCLOSURE OF THE INVENTION

In this method, however, the number of samples (e.g., cells) is limitedto at most about several dozen to detect FRET in a short period of time.In other words, it is difficult to statistically analyze a large numberof cells as analysis targets in a short period of time. Further, inconventional methods, only fluorescence intensity is measured. However,fluorescence intensity depends on the amount of fluorescent proteinlabel attached to a cell, which makes it impossible to completelyeliminate the uncertainties of measured fluorescence information. Forthis reason, such conventional methods have a problem that it isdifficult to quantitatively detect the degree of occurrence of FRET.

In order to solve the above problem, it is an object of the presentinvention to provide a method of and a device for detecting FRET capableof quantitatively measuring the degree of occurrence of FRET (e.g., FRETefficiency) of a sample containing a donor molecule and an acceptormolecule by eliminating the uncertainties of fluorescence detectioninformation resulting from the amount of fluorescent protein labelattached and by eliminating the uncertainties of fluorescence detectioninformation resulting from the broadening of wavelength range offluorescence emitted by the donor molecule and the broadening ofwavelength range of fluorescence emitted by the acceptor molecule.

In order to achieve the object, the present invention provides a FRETdetection method of detecting FRET (Fluorescence Resonance EnergyTransfer) in which energy of a first molecule is transferred to a secondmolecule.

The method includes the steps of:

A FRET detection method of detecting FRET (Fluorescence Resonance EnergyTransfer) in which energy of a donor molecule is transferred to anacceptor molecule, the method comprising the steps of:

a) measuring fluorescence emitted from each of samples by two or moredetection sensors having different light-receiving wavelength bands,each of the samples being labeled with a donor molecule and an acceptormolecule and being irradiated with laser light whose intensity ismodulated at a predetermined frequency, to acquire detection valuesincluding fluorescence intensity information and phase information onthe fluorescence emitted from each of the samples;

b) reading calibration information previously stored in a memory means,which includes at least a first intensity ratio that is a ratio betweenfluorescence intensities at the light-receiving wavelength bands of adonor molecule fluorescence component emitted from the donor moleculeincluded in the fluorescence emitted from each of the samples, phaseinformation on the donor molecule fluorescence component relative to themodulated laser light, a second intensity ratio that is a ratio betweenfluorescence intensities at the light-receiving wavelength bands of anacceptor molecule fluorescence component emitted from the acceptormolecule included in the fluorescence, phase information on the acceptormolecule fluorescence component relative to the laser light, and anon-FRET fluorescence lifetime of the donor molecule fluorescencecomponent when the FRET does not occur, which is a lifetime defined byassuming that fluorescence emitted from the donor molecule excited bylaser light is a relaxation response of a first-order lag system;

c) calculating fluorescence intensity information and phase informationon the fluorescence at each of the light-receiving wavelength bands,based on the detection values, and determining a FRET fluorescencelifetime of the donor molecule fluorescence component, which is definedby assuming that fluorescence emitted from the donor molecule excited bylaser light is a relaxation response of a first-order lag system, usingthe calculated fluorescence intensity information, the calculated phaseinformation, the first intensity ratio, the phase information on thedonor molecule fluorescence component, the second intensity ratio, thephase information on the acceptor molecule fluorescence component; and

d) determining information on FRET occurrence using a ratio between theFRET fluorescence lifetime of the donor molecule fluorescence componentand the non-FRET fluorescence lifetime of the donor moleculefluorescence component.

In the step d), FRET efficiency E_(t) is preferably determined as theinformation on the occurrence of FRET, which is represented by1−(τ_(d)*/τ_(d)), wherein τ_(d) is the non-FRET fluorescence lifetime ofthe donor molecule fluorescence component and τ_(d)* is the FRETfluorescence lifetime of the donor molecule fluorescence component.

Preferably, in the step c), fluorescence intensity information and phaseinformation on each of the samples are calculated based on each of thedetection values and the FRET fluorescence lifetime is determined basedon the calculated multiple pieces of fluorescence intensity informationand phase information.

Also preferably, in the step c), the fluorescence intensity informationand the phase information calculated at each of the light-receivingwavelength bands are represented as a vector, and

fluorescence intensity information and phase information on the donormolecule fluorescence component and fluorescence intensity informationand phase information on a FRET component of an acceptor moleculefluorescence component which is emitted by the donor molecule on theoccurrence of FRET are calculated at, and

the FRET fluorescence lifetime is determined by using the calculatedinformation.

And also preferably, the light-receiving wavelength bands include afirst wavelength band centered around a peak wavelength at which afluorescence intensity of the donor molecule fluorescence component ismaximum and a second wavelength band centered around a peak wavelengthat which a fluorescence intensity of the acceptor molecule fluorescencecomponent is maximum. In the step c), fluorescence intensity informationand phase information on the donor molecule fluorescence componentemitted are calculated using at least the second intensity ratio and thevector at the first wavelength band represented by the detection valuesacquired from the detection sensors with the first wavelength band, and

fluorescence intensity information and phase information on the FRETcomponent are calculated using at least the first intensity ratio andthe vector at the second wavelength band represented by the detectionvalues acquired from the detection sensors with the second wavelengthband.

Preferably, the memory means previously stores fluorescence intensityinformation and phase information on a directly-excited fluorescencecomponent of the acceptor molecule fluorescence component, thedirectly-excited fluorescence component being emitted by the acceptormolecule directly excited by the laser light,

in the step b), fluorescence intensity information and phase informationon the directly-excited fluorescence component are read from the memorymeans to represent the information on the directly-excited fluorescencecomponent as a vector, and

in the step c), fluorescence intensity information and phase informationon the acceptor molecule fluorescence component emitted at the time whenthe FRET occurs are calculated using at least the vector of thedirectly-excited fluorescence component and the vector at the secondwavelength band.

Also preferably, the memory means previously stores fluorescenceintensity information and phase information on a directly-excitedfluorescence component of the acceptor molecule fluorescence component,the directly-excited fluorescence component being emitted by theacceptor molecule directly excited by the laser light,

in the step b), fluorescence intensity information and phase informationon the directly-excited fluorescence component are read from the memorymeans and then represented as a vector of the directly-excitedfluorescence component, and

in the step c), phase information on the FRET component is furthercalculated using at least the vector at the second wavelength band andthe first intensity ratio, and

a FRET fluorescence lifetime of the acceptor molecule fluorescencecomponent emitted at the time when the FRET occurs and a non-FRETfluorescence lifetime of the acceptor molecule fluorescence componentemitted at the time when the FRET does not occur are determined usingthe calculated phase information on the FRET component and the vector ofthe directly-excited fluorescence component, and

a FRET fluorescence lifetime of the donor molecule fluorescencecomponent is determined using the FRET fluorescence lifetime of theacceptor molecule fluorescence component and the non-FRET fluorescencelifetime of the acceptor molecule fluorescence component.

Preferably, the method further includes the steps of:

preparing a predetermined sample which is each of the samples unlabeledwith the donor molecule and the acceptor molecule and emitsautofluorescence when irradiated with the laser light;

measuring, at each of the light-receiving wavelength bands, theautofluorescence emitted by the predetermined sample which is irradiatedwith the laser light;

calculating fluorescence intensity information and phase information onthe autofluorescence from the measured autofluorescence within each ofthe light-receiving wavelength bands to store the calculatedfluorescence intensity information and phase information in the memorymeans;

wherein

in the step b), the stored fluorescence intensity information and thephase information on the autofluorescence are read from the memory meansand the read fluorescence intensity information and the phaseinformation on the autofluorescence are represented as a vector, and

in the step c), the vector of the autofluorescence is subtracted fromeach vector at the light-receiving wavelength bands of the samples to bemeasured, and the FRET fluorescence lifetime is determined using avector obtained by the subtraction.

Also preferably, the autofluorescence is measured by each of thedetection sensors by irradiating the predetermined sample as a measuringobject with laser light whose intensity is modulated at a predeterminedfrequency.

Still also preferably, the method further includes the steps of:

preparing a non-FRET sample which is labeled with the donor molecule andthe acceptor molecule and which has been treated not to cause FRET;

measuring, at each of the light-receiving wavelength bands, fluorescenceemitted by the non-FRET sample which is irradiated with the laser light;and

calculating fluorescence intensity information and phase information onthe a directly-excited fluorescence component emitted by the acceptormolecule directly excited by laser light within each of thelight-receiving wavelength bands from the measured fluorescence, usingthe first intensity ratio previously stored in the memory means, thephase information on the donor molecule fluorescence component, thesecond intensity ratio previously stored in the memory means, and thephase information on the acceptor molecule fluorescence component, tostore the calculated fluorescence intensity information and phaseinformation in the memory means.

Still preferably, the non-FRET sample is a sample obtained by labeling,with the donor molecule and the acceptor molecule, a predeterminedsample which emits autofluorescence when excited by the laser light.

The method further includes the steps of:

preparing the predetermined sample;

measuring, at each of the light-receiving wavelength bands, theautofluorescence emitted by the predetermined sample which is irradiatedwith the laser light;

calculating fluorescence intensity information and phase information onthe autofluorescence from the measured autofluorescence within each ofthe light-receiving wavelength bands, to store the calculatedfluorescence intensity information and phase information on theautofluorescence in the memory means;

subtracting an autofluorescence vector representing the fluorescenceintensity information and phase information on the autofluorescence froma non-FRET sample vector representing fluorescence intensity informationand phase information on fluorescence emitted by the non-FRET sample;and

calculating a directly-excited fluorescence component vectorrepresenting the fluorescence intensity information and phaseinformation on the directly-excited fluorescence component, using avector obtained by the subtraction.

Preferably, the method further comprising the steps of:

preparing a donor molecule sample which is labeled with only the donormolecule;

measuring, at each of the light-receiving wavelength bands, fluorescenceemitted by the donor molecule of the donor molecule sample which isirradiated with the laser light;

calculating fluorescence intensity information and phase information onthe measured fluorescence emitted by the donor molecule sample withineach of the light-receiving wavelength bands, to obtain the firstintensity ratio; and

storing the phase information on the measured fluorescence and the firstintensity ratio.

More preferably, the donor molecule sample is a sample obtained bylabeling, with the donor molecule, a predetermined sample which emitsautofluorescence when excited by the laser light,

the method further comprising the steps of:

preparing the predetermined sample;

measuring, at each of the light-receiving wavelength bands, theautofluorescence emitted by the predetermined sample which is irradiatedwith the laser light;

calculating fluorescence intensity information and phase information onthe autofluorescence from the measured autofluorescence within each ofthe light-receiving wavelength bands to store the calculatedfluorescence intensity information and phase information in the memorymeans;

subtracting an autofluorescence vector representing the fluorescenceintensity information and phase information on the autofluorescence froma donor molecule sample vector representing fluorescence intensityinformation and phase information on fluorescence emitted by the donormolecule sample; and

calculating the fluorescence intensity information and phase informationon the fluorescence emitted by the donor molecule sample, using a vectorobtained by the subtraction.

And also preferably, the method further includes the steps of:

preparing an acceptor molecule sample which is labeled with only theacceptor molecule;

measuring, at each of the light-receiving wavelength bands, fluorescenceemitted by the acceptor molecule of the acceptor molecule sample whichis irradiated with the laser light;

calculating fluorescence intensity information and phase information onthe measured fluorescence emitted by the acceptor molecule sample withineach of the light-receiving wavelength bands, to obtain the secondintensity ratio; and

storing the phase information on the measured fluorescence and thesecond intensity ratio.

More preferably, the acceptor molecule sample is a sample obtained bylabeling, with the acceptor molecule, a predetermined sample which emitsautofluorescence when excited by the laser light.

The method further includes the steps of:

preparing the predetermined sample;

measuring, at each of the light-receiving wavelength bands, theautofluorescence emitted by the predetermined sample which is irradiatedwith the laser light;

calculating fluorescence intensity information and phase information onthe autofluorescence from the measured autofluorescence within each ofthe light-receiving wavelength bands, to store the calculatedfluorescence intensity information and phase information in the memorymeans;

subtracting an autofluorescence vector representing the fluorescenceintensity information and phase information on the autofluorescence froman acceptor molecule sample vector representing fluorescence intensityinformation and phase information on fluorescence emitted by theacceptor molecule sample; and

calculating the fluorescence intensity information and phase informationon the fluorescence emitted by the acceptor molecule sample, using avector obtained by the subtraction.

The present invention also provides a device for detecting FRET(Fluorescence Resonance Energy Transfer), in which energy of a firstmolecule transfers to a second molecule. The device includes:

an information acquiring unit which acquires detection values, eachvalues including fluorescence intensity information and phaseinformation on fluorescence emitted by each of the samples to bemeasured by allowing two or more detection sensors different inlight-receiving wavelength band to receive fluorescence emitted by eachof the samples to be measured, each of the samples being labeled with adonor molecule and an acceptor molecule and being irradiated with laserlight whose intensity is modulated at a predetermined frequency;

a memory means for previously storing calibration information includingat least a first intensity ratio that is a ratio between fluorescenceintensities at the light-receiving wavelength bands of a donor moleculefluorescence component emitted by the donor molecule included in thefluorescence emitted by each of the samples to be measured, phaseinformation on the donor molecule fluorescence component, an acceptorintensity ratio that is a ratio between fluorescence intensities at thelight-receiving wavelength bands of an acceptor molecule fluorescencecomponent emitted by the acceptor molecule, phase information of theacceptor molecule fluorescence component, and a non-FRET fluorescencelifetime, of the donor molecule fluorescence component emitted at thetime when the FRET does not occur, which is a lifetime defined byassuming that fluorescence emitted by the donor molecule excited bylaser light is a relaxation response of a first-order lag system;

a FRET fluorescence lifetime calculating unit which calculatesfluorescence intensity information and phase information on fluorescencewithin each of the light-receiving wavelength bands emitted by each ofthe samples to be measured based on the detection values acquired by thedetected information acquiring unit, and determines a FRET fluorescencelifetime of the donor molecule fluorescence component, which is definedby assuming that fluorescence emitted by the donor molecule excited bylaser light is a relaxation response of a first-order lag system, byusing the calculated fluorescence intensity information, the calculatedphase information, the first intensity ratio read from the memory means,the phase information on the donor molecule fluorescence component, thesecond intensity ratio, and the phase information on the acceptormolecule fluorescence component; and

a FRET occurrence information calculating unit which determinesinformation on the occurrence of FRET represented by a ratio between theFRET fluorescence lifetime of the donor molecule fluorescence componentand the non-FRET fluorescence lifetime of the donor moleculefluorescence component.

Preferably, in the FRET fluorescence lifetime calculating unit, thefluorescence intensity information and the phase information calculatedfrom each of the detection values are represented as a vector, andfluorescence intensity information and phase information on the donormolecule fluorescence component and fluorescence intensity informationand phase information on a FRET component of an acceptor moleculefluorescence component which is emitted by the acceptor molecule at thetime when the FRET occurs, are calculated using the vector, the firstintensity ratio, the phase information on the donor moleculefluorescence component, the second intensity ratio of the acceptormolecule fluorescence component, and the phase information on theacceptor molecule fluorescence component, and

the FRET fluorescence lifetime is determined by using the calculatedinformation.

Also preferably, the respective light-receiving wavelength bands of thesensors are a first wavelength band centered around a peak wavelength atwhich a fluorescence intensity of the donor molecule fluorescencecomponent is maximum and a second wavelength band centered around a peakwavelength at which a fluorescence intensity of the acceptor moleculefluorescence component is maximum, and

the FRET fluorescence lifetime calculating unit calculates fluorescenceintensity information and phase information on the donor moleculefluorescence component by using at least a vector at the firstwavelength band determined from the detection value acquired from one ofthe detection sensors with the first wavelength band and the secondintensity ratio, and calculates fluorescence intensity information andphase information on the FRET component by using at least a vector atthe second wavelength band represented by the detection value acquiredfrom one of the detection sensors with the second wavelength band andthe first intensity ratio.

Preferably, the device further includes an autofluorescence calibrationunit. The autofluorescence calibration unit acquires a detection valueincluding fluorescence intensity information and phase information ateach of the light-receiving wavelength bands from each of the detectionsensors by irradiating, with the laser, light a predetermined samplewhich is each of the samples unlabeled with the donor molecule and theacceptor molecule and emits autofluorescence when irradiated with thelaser light, calculates fluorescence intensity information and phaseinformation on the autofluorescence within each of the light-receivingwavelength bands, and stores the calculated fluorescence intensityinformation and the calculated phase information on the autofluorescencein the memory means, and

in the FRET fluorescence lifetime calculating unit, a vectorrepresenting the fluorescence intensity information and the phaseinformation on the autofluorescence is subtracted from a vectorrepresenting information on fluorescence within each of thelight-receiving wavelength bands emitted by the sample to be measured,and the FRET fluorescence lifetime is determined using a vector obtainedby the subtraction.

Preferably, the device further includes a non-FRET calibration unit. Thenon-FRET calibration unit calculates fluorescence intensity informationand phase information on fluorescence within each of the light-receivingwavelength bands emitted by a non-FRET sample, which has the donor andacceptor molecules attached thereto and which has been treated so as notto cause FRET, when a detection value including fluorescence intensityinformation and phase information, at each of the light-receivingwavelength bands is acquired from each of the detection sensors byirradiating the non-FRET sample with the laser light, and calculatesfluorescence intensity information and phase information on adirectly-excited fluorescence component emitted by the acceptor moleculedirectly excited by the laser light by using the calculated fluorescenceintensity information, the calculated phase information, the firstintensity ratio, the phase information on the donor moleculefluorescence component, the second intensity ratio, and the phaseinformation on the acceptor molecule fluorescence component, and derivesa directly-excited fluorescence component vector representing thecalculated information, and stores a derived result in the memory means,and

the FRET fluorescence lifetime calculating unit determines the FRETfluorescence lifetime by using the directly-excited fluorescencecomponent vector.

Preferably, the device further includes a donor molecule calibrationunit. The donor molecule calibration unit calculates fluorescenceintensity information and phase information on fluorescence within eachof the light-receiving wavelength bands emitted by a donor moleculesample, which is each of the samples labeled with only the donormolecule, when the donor molecule sample irradiated with the laser lightemits fluorescence and a detection value including fluorescenceintensity information and phase information on the fluorescence withineach of the light-receiving wavelength bands emitted by the donormolecule sample is acquired from each of the detection sensors, andcalculates the first intensity ratio, and stores the calculated firstintensity ratio and the calculated phase information on fluorescenceemitted by the donor molecule sample in the memory means.

Preferably, the device further includes an acceptor molecule calibrationunit. The acceptor molecule calibration unit calculates fluorescenceintensity information and phase information on fluorescence within eachof the light-receiving wavelength bands emitted by an acceptor moleculesample, which is each of the samples labeled with only the acceptormolecule, when the acceptor molecule sample irradiated with the laserlight emits fluorescence and a detection value including fluorescenceintensity information and phase information on the fluorescence withineach of the light-receiving wavelength bands emitted by the acceptormolecule sample is acquired from each of the detection sensors, andcalculates, the second intensity ratio, and stores the calculated secondintensity ratio and the calculated phase information on fluorescenceemitted by the acceptor molecule sample in the memory means.

EFFECTS OF THE INVENTION

According to the present invention, it is possible to provide a methodof and a device for detecting FRET capable of quantitatively measuringthe degree of occurrence of FRET (e.g., FRET efficiency) of a sample tobe measured, which is obtained by, for example, attaching a donormolecule (fluorescent protein) and an acceptor molecule (fluorescentprotein) to a cell, by eliminating the uncertainties of fluorescencedetection information resulting from the amount of fluorescent proteinlabel attached and by eliminating the uncertainties of fluorescencedetection information resulting from the overlap between thelight-receiving wavelength bands of fluorescence emitted by the donormolecule and the wavelength band of fluorescence emitted by the acceptormolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic configuration diagram of a flow cytometer using aFRET detection device according to the present invention.

FIG. 2 is a chart showing examples of the energy absorption spectrum andfluorescence spectrum of a donor molecule and examples of the energyabsorption spectrum and fluorescence spectrum of an acceptor molecule.

FIG. 3 is a schematic diagram illustrating the occurrence of FRET.

FIG. 4 is a schematic configuration diagram of one example of alight-receiving unit of the flow cytometer shown in FIG. 1.

FIG. 5 is a diagram illustrating the components of fluorescence emittedby a FRET sample and the incidence of these fluorescence components intophotoelectric converters in the flow cytometer shown in FIG. 1.

FIG. 6 is a schematic configuration diagram of one example of a controland processing section of the flow cytometer shown in FIG. 1.

FIG. 7 is a schematic configuration diagram of one example of ananalysis device of the flow cytometer shown in FIG. 1.

FIG. 8 is a diagram illustrating a model of dynamics of fluorescenceemission at the time when FRET occurs.

FIG. 9 is a diagram illustrating the components of fluorescence emittedby an unlabeled sample and the incidence of these fluorescencecomponents into photoelectric converters in the flow cytometer shown inFIG. 1.

FIG. 10 is a diagram illustrating the components of fluorescence emittedby a donor-labeled sample and the incidence of these fluorescencecomponents into photoelectric converters in the flow cytometer shown inFIG. 1.

FIG. 11 is a diagram illustrating the components of fluorescence emittedby an acceptor-labeled sample and the incidence of these fluorescencecomponents into photoelectric converters in the flow cytometer shown inFIG. 1.

FIG. 12 is a diagram illustrating the components of fluorescence emittedby a non-FRET labeled sample and the incidence of these fluorescencecomponents into photoelectric converters in the flow cytometer shown inFIG. 1.

FIG. 13 is a flowchart of a FRET detection method carried out by theflow cytometer shown in FIG. 1.

FIG. 14 is a flowchart of first calibration carried out by the flowcytometer shown in FIG. 1.

FIG. 15 is a flowchart of second calibration carried out by the flowcytometer shown in FIG. 1.

FIG. 16 is a flowchart of third calibration carried out by the flowcytometer shown in FIG. 1.

FIG. 17 is a flowchart of fourth calibration carried out by the flowcytometer shown in FIG. 1.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, a method of and a device for detecting FRET according tothe present invention will be described in detail based on a flowcytometer.

FIG. 1 is a schematic configuration diagram of a flow cytometer 10 usinga FRET detection device according to the present invention.

The flow cytometer 10 mainly includes a signal processing device 20 andan analysis device (computer) 80.

The signal processing device 20 irradiates a FRET detection targetsample 12 (hereinafter, referred to as a “FRET sample 12”) with laserlight and detects and processes a fluorescence signal of fluorescenceemitted by the FRET sample 12. The FRET sample 12 is obtained bylabeling a receptor sample such as a specific cell (hereinafter,referred to as a “cell”) with a donor molecule and an acceptor moleculeby chemical or physical bonding. The analysis device 80 analyzes theFRET sample 12 based on a processing result obtained from the signalprocessing device 20. The sample emits autofluorescence when irradiatedwith laser light. Such autofluorescence becomes a measurement noisecomponent. Therefore, in fact, it is preferred that no autofluorescencebe generated. However, a certain degree of autofluorescence isgenerated, for example, in a case where a cell or the like is irradiatedwith laser light.

The flow cytometer 10 is one embodiment of the present invention. Theflow cytometer 10 is a device in which a plurality of FRET samplessuspended in a measurement solution are irradiated with laser light, andthe respective fluorescence lifetimes (fluorescence relaxation timeconstants) of fluorescence components (a fluorescence component within adonor wavelength band and a fluorescence component within an acceptorwavelength band, which will be described later) of fluorescence emittedby each of the FRET samples are calculated to determine FRET efficiencyindicating the degree of occurrence of FRET of the FRET sample.

The signal processing device 20 includes a laser light source unit 22,light-receiving units 24 and 26, a control and processing section 28,and a tube line 30. The control and processing section 28 includes acontrol unit and a processing unit. The control unit modulates theintensity of laser light to be emitted from the laser light source unit22 at a predetermined frequency. The processing unit processes afluorescence signal from the FRET sample 12 that flows through the tubeline 30 with a sheath liquid that forms a high-speed flow, therebyforming a flow cell.

A recovery container 32 is disposed at the outlet of the tube line 30.The flow cytometer 10 may include a cell sorter for separating a livingsubstance (e.g., specific cells) contained in the FRET samples 12 withina short period of time by irradiation with laser light to recover theliving substance in a different recovery container.

The laser light source unit 22 is a light source that emits laser lightto excite the donor molecule. For example, in a case where CFP (CyanFluorescent Protein) is used as the donor molecule and YFP (YellowFluorescent Protein) is used as the acceptor molecule, laser lighthaving a wavelength of 405 to 440 nm is used to mainly excite the donormolecule. More specifically, the laser light source unit 22 is a unitthat emits laser light having a wavelength to excite the donor moleculeunder the condition that its intensity is modulated at a predeterminedfrequency.

Here, the occurrence of FRET is briefly explained. FIG. 2 is a chartshowing examples of the energy absorption spectrum and fluorescencespectrum of the donor molecule and examples of the energy absorptionspectrum and fluorescence spectrum of the acceptor molecule. FIG. 3 is adiagram illustrating the occurrence of FRET in an easily understandablemanner.

FIG. 2 shows the characteristics of energy absorption and fluorescenceemission of the donor and acceptor molecules. In this case, the donormolecule is CFP (Cyan Fluorescent Protein) and the acceptor molecule isYFP (Yellow Fluorescent. Protein). In FIG. 2, curve A₁ indicates theenergy absorption spectrum of CFP, curve A₂ indicates the fluorescenceemission spectrum of CFP, curve B₁ indicates the energy absorptionspectrum of YFP, and curve B₂ indicates the fluorescence emissionspectrum of YFP. In FIG. 2, a hatched area indicates a wavelength bandwhere YFP absorbs the energy of fluorescence emitted by CFP so that FREToccurs.

Generally, FRET occurs by the following mechanism. A donor molecule isexcited by laser light, part of the excited donor molecule emitsfluorescence, and the energy of the excited donor molecule is partiallytransferred to an acceptor molecule by the coulomb interaction. Suchenergy transfer occurs between positions spaced away by a very smalldistance of 2 nm or less, and indicates interaction (bonding) betweenmolecules. When the energy transfer occurs, the acceptor molecule isexcited and emits fluorescence. In order to allow FRET to occur, asshown in FIG. 2, it is necessary for the fluorescence emissionwavelength band of the donor molecule and the energy absorptionwavelength band of the acceptor molecule to partially overlap eachother.

Generally, in a molecule, the peak wavelength of its energy absorptionspectrum and the peak wavelength of its fluorescence spectrum are veryclose to each other unless its fluorescence emission energy issignificantly large. As shown in FIG. 2, the same applies to CFP (CyanFluorescent Protein) as the donor molecule and to YFP (YellowFluorescent Protein) as the acceptor molecule. As described above, inorder to allow FRET to occur, it is necessary for the fluorescenceemission wavelength band of the donor molecule and the energy absorptionwavelength band of the acceptor molecule to partially overlap eachother. Therefore, the peak wavelengths of the curves A₁, A₂, B₁, and B₂shown in FIG. 2 are of course concentrated in a narrow wavelength bandand these wavelength spectra partially overlap one another. Therefore,in a case where laser light (e.g., laser light modulated at a frequencyf) is emitted from the laser light source unit 22 to excite the donormolecule, the acceptor molecule is also directly excited by the laserlight so that fluorescence is emitted not only from the donor moleculebut also from the acceptor molecule (see FIG. 3).

In the flow cytometer 10, laser light is emitted from the laser lightsource unit 22, and as a result, the FRET sample 12 to be measured emitsautofluorescence (not shown in FIG. 3) emitted by the cell containedtherein, fluorescence emitted by the excited donor molecule containedtherein, fluorescence emitted by the excited acceptor molecule containedtherein, and FRET fluorescence emitted by the acceptor molecule.

The light-receiving unit 24 is disposed so as to be opposite to thelaser light source unit 22 with the tube line 30 interposedtherebetween. The light-receiving unit 24 is provided with aphotoelectric converter. In response to laser light forwardly scatteredby the FRET sample 12 passing through a measurement point in the tubeline 30, the photoelectric converter outputs a detection signalindicating that the FRET sample 12 is passing through the measurementpoint. The signal output from the light-receiving unit 24 is supplied tothe control and processing section 28, in which the signal is used as atrigger signal indicating the timing of passage of the FRET sample 12through the measurement point.

On the other hand, the light-receiving unit 26 is disposed so as to beorthogonal to a direction in which laser light is emitted from the laserlight source unit 22 and also orthogonal to a direction in which theFRET sample 12 moves in the tube line 30. The light-receiving unit 26 isprovided with a photoelectric converter. The photoelectric converterreceives fluorescence emitted by the FRET sample 12 irradiated at themeasurement point. A photomultiplier and an avalanche photodiode areexamples of the photoelectric converter.

FIG. 4 is a schematic configuration diagram schematically showing theconfiguration of one example of the light-receiving unit 26. Thelight-receiving unit 26 shown in FIG. 4 includes a lens system 26 a,dichroic mirrors 26 b ₁ and 26 b ₂, band-pass filters 26 c ₁ and 26 c ₂,and photoelectric converters 27 a and 27 b. The lens system 26 a focusesa fluorescence signal of fluorescence emitted by the FRET sample 12. Asthe photoelectric converters 27 a and 27 b, photomultipliers, avalanchephotodiodes, or the like are used. The lens system 26 a is configured tofocus fluorescence received by the light-receiving unit 26 on thelight-receiving surfaces of the photoelectric converters 27 a and 27 b.

The dichroic mirrors 26 b ₁ and 26 b ₂ reflect fluorescence within apredetermined wavelength band but transmits other wavelengths offluorescence. The reflection wavelength band and transmission wavelengthband of the dichroic mirror 26 b ₁ are set so that, after filtering bythe band-pass filter 26 c ₁, fluorescence within a predeterminedwavelength band can be introduced into the photoelectric converter 27 a.Also, the reflection wavelength band and transmission wavelength band ofthe dichroic mirror 26 b ₂ are set so that, after filtering by theband-pass filter 26 c ₂, fluorescence within a predetermined wavelengthband can be introduced into the photoelectric converter 27 b.

The band-pass filter 26 c ₁ is a filter disposed in front of thelight-receiving surface of the photoelectric converter 27 a andtransmits only fluorescence within a predetermined wavelength band.Also, the band-pass filter 26 c ₂ is a filter disposed in front of thelight-receiving surface of the photoelectric converter 27 b andtransmits only fluorescence within a predetermined wavelength band. Thefluorescence wavelength bands that the filters 26 c ₁ and 26 c ₂transmit are set so as to correspond to the wavelength band offluorescence to be emitted, and are herein different from each other.

In a case where CFP (Cyan Fluorescent Protein) is used as the donormolecule and YFP (Yellow Fluorescent Protein) is used as the acceptormolecule, the fluorescence wavelength bands that the filters 26 c ₁ and26 c ₂ transmit are set so that fluorescence emitted by the donormolecule (indicated by the curve A₂ in FIG. 2) and fluorescence emittedby the acceptor molecule (indicated by the curve B₂ in FIG. 2) can beseparately measured. For example, a wavelength band indicated by anarrow A in FIG. 2 is set as a wavelength band (donor wavelength band)that one of the filters transmits so that fluorescence emitted by thedonor molecule (donor fluorescence) is mainly transmitted, and awavelength band indicated by an arrow B in FIG. 2 is set as a wavelengthband (acceptor wavelength band) that the other filter transmits so thatfluorescence emitted by the acceptor molecule (acceptor fluorescence) ismainly transmitted.

Here, as described above, the wavelength band of the donor fluorescenceand the wavelength band of the acceptor fluorescence actually overlapeach other, and therefore, transmitted light (fluorescence) within thedonor wavelength band contains a leaked acceptor fluorescence component.Also, transmitted light (fluorescence) within the acceptor wavelengthband contains a leaked donor fluorescence component. It can be said thatsuch leakage inevitably occurs under conditions where FRET can occur(i.e., under conditions where the fluorescence emission wavelength bandof the donor molecule and the energy absorption wavelength band of theacceptor molecule partially overlap each other). In conventional methodsfor measuring and evaluating the occurrence of FRET, such leakedcomponents are uncertainties that reduce the accuracy of measurement andevaluation. However, the present invention achieves high-accuracymeasurement of FRET and high-accuracy evaluation of FRET based onmeasurement results by taking the influence of such leaked componentsinto consideration.

The photoelectric converters 27 a and 27 b are each a sensor thatconverts light received by its photoelectric surface into an electricsignal. Each of the photoelectric converters 27 a and 27 b is providedwith a sensor including, for example, a photomultiplier. Thelight-receiving unit 26 is configured so that the photoelectricconverter 27 a can receive light within the donor wavelength band andthe photoelectric converter 27 b can receive light within the acceptorwavelength band.

FIG. 5 is a schematic diagram illustrating fluorescence componentsemitted by the FRET sample 12, which is irradiated with laser lightemitted from the laser light source unit 22 while flowing through thetube line 30, and the incidence of these fluorescence components intothe light-receiving surfaces of the photoelectric converters 27 a and 27b. The FRET sample 12 is a sample in which CFP (Cyan FluorescentProtein) as the donor molecule and YFP (Yellow Fluorescent Protein) asthe acceptor molecule are attached to a cell that emitsautofluorescence. When laser light is emitted from the laser lightsource unit 22, the FRET sample 12 that flows through the tube line 30emits fluorescence f_(d), fluorescence f_(a), fluorescence f_(s), andfluorescence f_(f). The fluorescence f_(d) is fluorescence emitted bythe donor molecule contained in the FRET sample 12 and directly excitedby the laser light. The fluorescence f_(a) is fluorescence emitted bythe acceptor molecule contained in the FRET sample 12 and directlyexcited by the laser light. The fluorescence f_(s) is autofluorescenceemitted by the cell contained in the FRET sample 12. The fluorescencef_(f) is FRET fluorescence emitted by the acceptor molecule. Each of thefluorescences is separated by the mirrors and the filters into a donorwavelength band component and an acceptor wavelength band component, andthe donor wavelength band components and the acceptor wavelength bandcomponents of these fluorescences are introduced into the photoelectricconverters 27 a and 27 b, respectively. Here, the FRET fluorescencef_(f) is weaker than other fluorescences emitted by direct excitation bylaser, and therefore it can be assumed that its donor wavelength bandcomponent (leaked component) is sufficiently small.

Light received by the photoelectric converter 27 a containsfluorescences indicated by routes R₁ to R₃ in FIG. 5, and light receivedby the photoelectric converter 27 b contains fluorescences indicated byroutes R₄ to R₇ in FIG. 5. Processing performed by each of theabove-mentioned units of the flow cytometer 10 will be described below,which is performed on both the light (fluorescence within the donorwavelength band) received by the photoelectric converter 27 a and thelight (fluorescence within the acceptor wavelength band) received by thephotoelectric converter 27 b.

Each of the photoelectric converters 27 a and 27 b receives light as anoptical signal having signal information, and therefore an electricsignal output from each of the photoelectric converters 27 a and 27 b isa fluorescence signal having signal information about phase difference.The fluorescence signal is supplied to the control and processingsection 28, and is then amplified by an amplifier and sent to theanalysis device 80.

As shown in FIG. 6, the control and processing section 28 includes asignal generation unit 40, a signal processing unit 42, and a controller44. The signal generation unit 40 and the controller 44 constitute alight source control unit that generates a modulation signal having apredetermined frequency.

The signal generation unit 40 is a unit that generates a modulationsignal for modulating (amplitude modulating) the intensity of laserlight at a predetermined frequency.

More specifically, the signal generation unit 40 includes an oscillator46, a power splitter 48, and amplifiers 50 and 52, and suppliesgenerated modulation signals to the laser light source unit 22 and alsoto the signal processing unit 42. As will be described later, themodulation signal is supplied to the signal processing unit 42 and isused as a reference signal for detecting the phase difference offluorescence signals output from the photoelectric converters 27 a and27 b. It is to be noted that the modulation signal is a sinusoidal wavesignal having a predetermined frequency. In this case, the frequency isset in the range of 10 to 100 MHz.

The signal processing unit 42 extracts, by using fluorescence signalsoutput from the photoelectric converters 27 a and 27 b, informationabout the phase delay (phase difference) of fluorescence emitted fromthe FRET sample 12 by irradiation with laser light. The signalprocessing unit 42 includes amplifiers 54 a and 54 b and a phasedifference detector 56 having a power splitter (not shown) and IQ mixers(not shown). The amplifiers 54 a and 54 b amplify fluorescence signalsoutput from the photoelectric converters 27 a and 27 b. The powersplitter (not shown) splits the modulation signal, which is a sinusoidalwave signal supplied from the signal generation unit 40, into splitsignals and provides the split signals to the amplified fluorescencesignals, respectively. Each of the IQ mixers (not shown) mixes theamplified fluorescence signal with the modulation signal.

One of the IQ mixers (not shown) of the phase difference detector 56 isprovided to mix the fluorescence signal supplied from the photoelectricconverter 27 a with the modulation signal supplied from the signalgeneration unit 40 as a reference signal. The other IQ mixer (not shown)of the phase difference detector 56 is provided to mix the fluorescencesignal supplied from the photoelectric converter 27 b with themodulation signal supplied from the signal generation unit 40 as areference signal. More specifically, each of the IQ mixers multipliesthe reference signal by the fluorescence signal (RF signal) to calculatea processing signal including the cos component (real part) and thehigh-frequency component of the fluorescence signal. Simultaneously,each of the IQ mixers also multiplies a signal, which is obtained byshifting the phase of the reference signal by 90 degrees, by thefluorescence signal to calculate a processing signal including the sincomponent (imaginary part) and the high-frequency component of thefluorescence signal. The processing signal including the cos componentand the processing signal including the sin component are supplied tothe controller 44.

The controller 44 is a unit that controls the signal generation unit 40to generate a sinusoidal wave signal having a predetermined frequency.The controller 44 is also a unit that obtains the cos component and thesin component of the fluorescence signal by removing the high-frequencycomponent from the processing signals which are obtained by the signalprocessing unit 42 and which include the cos component and the sincomponent of the fluorescence signal.

More specifically, the controller 44 includes a system controller 60, alow-pass filter 62, an amplifier 64, and an A/D converter 66. The systemcontroller 60 gives instructions for controlling the respectiveoperations of the units and manages all the operations of the flowcytometer 10. The low-pass filter 62 removes the high-frequencycomponent from the processing signal in which the high-frequencycomponent is added to the cos component and from the processing signalin which the high-frequency component is added to the sin component.Here, the processing signals are calculated by the signal processingunit 42. The amplifier 64 amplifies the processing signal including thecos component which is obtained by removing the high-frequency componentand the processing signal including the sin component which is obtainedby removing the high-frequency component. The A/D converter 66 samplesthe amplified processing signals. In the A/D converter 66, theprocessing signal including the cos component which is obtained byremoving the high-frequency component and the processing signalincluding the sin component which is obtained by removing high-frequencycomponent are sampled and then supplied to the analysis device 80.

The analysis device 80 is a device that calculates, from the processingsignal values (detection values) of the cos component (real part) andthe sin component (imaginary part) of the fluorescence signal,fluorescence intensity information and phase information on eachfluorescence component, fluorescence lifetime (fluorescence relaxationtime constant), and FRET efficiency. For example, the analysis device 80calculates the fluorescence intensity P_(d) and phase θ_(d) (measurementvalues) of fluorescence within the donor wavelength band from adetection value obtained by the photoelectric converter 27 a, andcalculates the fluorescence intensity P_(a) and phase θ_(a) (measurementvalues) of fluorescence within the acceptor wavelength band from adetection value obtained by the photoelectric converter 27 b. Theanalysis device 80 corresponds to the FRET detection device according tothe present invention and implements a FRET detection method which willbe described later.

FIG. 7 is a schematic configuration diagram of the analysis device 80.

The analysis device 80 is a device that is configured to execute apredetermined program on a computer. The analysis device 80 includes, inaddition to a CPU 82, a memory 84, and an input/output port 86, a firstcalibration unit 88, a second calibration unit 90, a third calibrationunit 92, a fourth calibration unit 94, a fluorescence relaxation timeconstant calculating unit 96, and a FRET efficiency calculating unit 98which are formed by executing software. The analysis device 80 isconnected with a display 94.

The CPU 82 is a calculating processor provided in the computer, andsubstantially executes various calculations required by the firstcalibration unit 88, the second calibration unit 90, the thirdcalibration unit 92, the fourth calibration unit 94, the fluorescencerelaxation time constant calculating unit 96, and the FRET efficiencycalculating unit 98. The first calibration unit 88 performsautofluorescence calibration, the second calibration unit 90 performsfirst molecule calibration, the third calibration unit 92 performssecond molecule calibration, and the fourth calibration unit 94 performsnon-FRET calibration.

The memory 84 includes a ROM that stores the program executed on thecomputer to form the first calibration unit 88, the second calibrationunit 90, the third calibration unit 92, the fourth calibration unit 94,the fluorescence relaxation time constant calculating unit 96, and theFRET efficiency calculating unit 98 and a RAM that stores processingresults calculated by these units and data supplied from theinput/output port 86.

The input/output port 86 is used to accept the input of detection valuesof the cos component (real part) and the sin component (imaginary part)of the fluorescence signal supplied from the controller 44 and also tooutput information such as the values of processing results calculatedby the units 88, 90, 92, 94, 96, and 98 or a scatter diagram onto thedisplay 94. The display 94 displays amplitude information and phasedifference information on fluorescence or the values of processingresults, such as fluorescence relaxation time constant and FRETefficiency, determined by the units 88, 90, 92, 94, 96, and 98 or agraph such as a scatter diagram.

The fluorescence relaxation time constant calculating unit 96calculates, from the detection values of the cos component and the sincomponent supplied from the controller 44, fluorescence intensityinformation and phase information (information about phase difference)on fluorescence received by each of the photoelectric converters 27 aand 27 b. Then, a FRET fluorescence lifetime of donor moleculefluorescence component, a FRET fluorescence lifetime of acceptormolecule fluorescence component, and a non-FRET fluorescence lifetime ofacceptor molecule fluorescence component are determined using thecalculated fluorescence intensity information, the calculated phaseinformation, and calibration information (which will be described later)previously stored in the memory 84. The FRET fluorescence lifetime ofdonor molecule fluorescence component refers to a fluorescence lifetimeof a fluorescence component emitted by the donor molecule excited bylaser light at the time when FRET occurs. The FRET fluorescence lifetimeof acceptor molecule fluorescence component refers to a fluorescencelifetime of a fluorescence component emitted by the acceptor moleculeexcited by laser light at the time when FRET occurs. The non-FRETfluorescence lifetime of acceptor molecule fluorescence component refersto a fluorescence lifetime of a fluorescence component emitted by theacceptor molecule excited by laser light at the time when FRET does notoccur. Each fluorescence lifetime is expressed as a fluorescencerelaxation time constant defined by assuming that all the fluorescencecomponents emitted by the FRET sample 12 irradiated with laser light arebased on relaxation responses of first-order lag system. Thefluorescence relaxation time constant calculating unit 96 will bedescribed later in detail.

The FRET efficiency calculating unit 98 determines FRET efficiencyrepresented by the ratio between the FRET fluorescence lifetime of donormolecule fluorescence component and a non-FRET fluorescence lifetime ofdonor molecule fluorescence component, both of the lifetimes determinedby the fluorescence relaxation time constant calculating unit 96. Thenon-FRET fluorescence lifetime of donor molecule fluorescence componentis included in the calibration information. The non-FRET fluorescencelifetime of donor molecule fluorescence component refers to afluorescence lifetime of a fluorescence component emitted by the donormolecule excited by laser light at the time when FRET does not occur.

The FRET efficiency calculating unit 98 may determine FRET efficiency byusing the FRET fluorescence lifetime of acceptor molecule fluorescencecomponent and the non-FRET fluorescence lifetime of acceptor moleculefluorescence component determined by the fluorescence relaxation timeconstant calculating unit 96. In this case, the value of the FRETefficiency is different from that of the above case. The FRET efficiencycalculating unit 98 will be described later in detail.

Hereinbelow, the principles of the FRET detection method according tothe present invention will be described. As described above, when theFRET sample having the donor molecule and the acceptor molecule isirradiated with laser light to mainly excite the donor molecule and thenFRET occurs, the labeled sample emits fluorescence f_(d), fluorescencef_(f), fluorescence f_(a), and fluorescence f_(s) shown in FIG. 5.

First, consideration is given to a fluorescence emission model,including a non-radiative process, at the time when the FRET sample isexcited by an ideal pulse light source with a pulse width of 0. Animpulse response (the number of photons emitted per unit volume(fluorescence intensity)) can be represented by the following formula(1). When a fluorescent molecule absorbs light, it takes only 10⁻¹⁵second for electrons in the fluorescent molecule to transit to theexcited state. Then, after a lapse of 10⁻¹³ to 10⁻¹¹ second, theelectrons fall from the first excited state by intramolecular relaxationprocess. Therefore, as in the model represented by the following formula(1), the dynamics of such absorption and intramolecular relaxationprocesses are neglected, and consideration is herein given to only theprocess of radiative transition from the first excited state.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 1} \right\rbrack & \; \\{{F(t)} = {{k_{f}{N(t)}} = {{k_{f}N_{0}{\mathbb{e}}^{{- {({k_{f} + k_{nf}})}}t}} = {\frac{N_{0}}{\tau_{0}}{\mathbb{e}}^{{- t}/\tau}}}}} & (1)\end{matrix}$

wherein

N(t): number of fluorescent molecules in excited state per unit volume(excited-state density function)

N₀: number of fluorescent molecules in excited state at time 0 per unitvolume

k_(f): rate constant of radiative transition (ratio of number ofmolecules undergoing radiative transition per unit time)

k_(nf): rate constant of non-radiative transition (ratio of number ofmolecules undergoing non-radiative transition per unit time)

τ₀≡1/k_(f): lifetime of excited state defined by assuming that there isno non-radiative process (natural lifetime)

τ≡1/(k_(f)+k_(nf)): fluorescence relaxation time constant

Here, N₀ depends on the molar absorbance coefficient and molarconcentration of the fluorescent molecules, and therefore variesdepending on the amount of fluorescent molecule label attached (i.e.,depending on the amount or state of the donor molecule or the acceptormolecule attached to the cell). It is to be noted that k_(f) and k_(nf)have a relationship represented by the following formula (2), wherein φrepresents the quantum yield of the fluorescent molecule.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 2} \right\rbrack & \; \\{\frac{k_{f}}{k_{f} + k_{nr}} = \phi} & (2)\end{matrix}$

The differential equation of a system whose impulse response isrepresented by the formula (1) can be represented by the followingformula when the incident power of laser is defined as u(t).

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 3} \right\rbrack & \; \\{\frac{\mathbb{d}{N(t)}}{\mathbb{d}t} = {{{- \left( {k_{f} + k_{nr}} \right)}{N(t)}} + {N_{0}{u(t)}}}} & (3)\end{matrix}$

Based on such an impulse model of fluorescence emission from thefluorescent molecule, consideration is given to a dynamics model offluorescence emission process of each of the fluorescence componentsshown in FIG. 5 generated when FRET occurs. When FRET occurs in the FRETsample having the donor molecule and the acceptor molecule, radiativetransition, non-radiative transition, and excitation energy transferproceed concurrently one another in the donor molecule. The model of afluorescence component within the donor wavelength band emitted by thedonor molecule (corresponding to the route R₁ shown in FIG. 5) can berepresented by the following formulas (4) and (5).

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 4} \right\rbrack & \; \\{\frac{\mathbb{d}{N_{d}(t)}}{\mathbb{d}t} = {{{- \left( {k_{d} + k_{t}} \right)}{N_{d}(t)}} + {N_{d\; 0}{u(t)}}}} & (4) \\{{F_{d}(t)} = {k_{df}{N_{d}(t)}}} & (5)\end{matrix}$

On the other hand, the acceptor molecule in the excited state isadditionally excited by excitation energy transfer. The model offluorescence components within the acceptor wavelength band emitted bythe acceptor molecule (corresponding to the routes R₄ and R₇ shown inFIG. 5) can be represented by the following formulas (6) and (7).

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 5} \right\rbrack & \; \\{\frac{\mathbb{d}{N_{a}(t)}}{\mathbb{d}t} = {{k_{t}{N_{d}(t)}} - {k_{a}{N_{a}(t)}} + {N_{a\; 0}{u(t)}}}} & (6) \\{{F_{a}(t)} = {k_{af}{N_{a}(t)}}} & (7)\end{matrix}$

wherein N_(d)(t) represents the number of donor fluorescent molecules inthe excited state per unit volume, N_(p)(t) represents the number ofacceptor fluorescent molecules in the excited state per unit volume,k_(d) represents the sum of the rate constant of radiative transitionand the rate constant of non-radiative transition of the donor molecule,k_(a) represents the sum of the rate constant of radiative transitionand the rate constant of non-radiative transition of the acceptormolecule, k_(df) represents the rate constant of radiative transition ofthe donor molecule, k_(af) represents the rate constant of radiativetransition of the acceptor molecule, N_(d0) represents the number ofdonor fluorescent molecules in the excited state at time 0 per unitvolume, N_(a0) represents the number of acceptor fluorescent moleculesin the excited state at time 0 per unit volume, and k_(t) represents therate constant of energy transfer from the donor molecule to the acceptormolecule. It is to be noted that the term k_(t)N_(d)(t) in the formula(7) corresponds to the fluorescence component indicated by the route R₇in FIG. 5, which is emitted by the acceptor molecule by FRET.

Further, the model of a fluorescence component within the acceptorwavelength band emitted by the donor molecule (corresponding to theroute R₅ shown in FIG. 5) can be represented by the following formulas(8) and (9). The model of a fluorescence component within the donorwavelength band emitted by the acceptor molecule (corresponding to theroute R₂ shown in FIG. 5) can be represented by the following formulas(10) and (11). It is to be noted that in general, it can also beconsidered that the dynamics of fluorescence of the donor molecule andthe dynamics of fluorescence of the acceptor molecule are not changeddepending on the wavelength band chosen, but here, it is more generallyassumed that the dynamics of fluorescence of the donor molecule and thedynamics of fluorescence of the acceptor molecule are changed dependingon the wavelength band chosen.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 6} \right\rbrack & \; \\{\frac{\mathbb{d}{N_{da}(t)}}{\mathbb{d}t} = {{{- k_{da}}{N_{da}(t)}} + {N_{{da}\; 0}{u(t)}}}} & (8) \\{{F_{da}(t)} = {k_{daf}{N_{da}(t)}}} & (9) \\{\frac{\mathbb{d}{N_{ad}(t)}}{\mathbb{d}t} = {{{- k_{ad}}{N_{ad}(t)}} + {N_{{ad}\; 0}{u(t)}}}} & (10) \\{{F_{ad}(t)} = {k_{adf}{N_{ad}(t)}}} & (11)\end{matrix}$

wherein N_(da)(t) represents the number of donor fluorescent moleculesin the excited state per unit volume within the acceptor wavelengthband, N_(ad)(t) represents the number of acceptor fluorescent moleculesin the excited state per unit volume within the donor wavelength band,k_(da) represents the sum of the rate constant of radiative transitionand the rate constant of non-radiative transition of the donor moleculewithin the acceptor wavelength band, k_(ad) represents the sum of therate constant of radiative transition and the rate constant ofnon-radiative transition of the acceptor molecule within the donorwavelength band, k_(daf) represents the rate constant of radiativetransition of the donor molecule within the acceptor wavelength band,k_(adf) represents the rate constant of radiative transition of theacceptor molecule within the donor wavelength band, N_(da0) representsthe number of donor fluorescent molecules in the excited state at time 0per unit volume within the acceptor wavelength band, and N_(ad0)represents the number of acceptor fluorescent molecules in the excitedstate at time 0 per unit volume within the donor wavelength band.

The model of a fluorescence component within the donor wavelength bandof autofluorescence emitted by the cell (corresponding to the route R₃shown in FIG. 5) can be represented by the following formulas (12) and(13). The model of a fluorescence component within the acceptorwavelength band of autofluorescence emitted by the cell (correspondingto the route R₆ shown in FIG. 5) can be represented by the followingformulas (14) and (15). It can be considered that various kinds ofmolecules contribute to the emission of autofluorescence from the cell,and therefore the model of autofluorescence may be different from themodel of a single emission of fluorescence. However, in general,autofluorescence is not strong, and therefore, in the following formulas(12) to (15), the characteristics of the sum of fluorescences emitted bythe various kinds of molecules are first-order approximated.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 7} \right\rbrack & \; \\{\frac{\mathbb{d}{N_{bd}(t)}}{\mathbb{d}t} = {{{- k_{bd}}{N_{bd}(t)}} + {N_{{bd}\; 0}{u(t)}}}} & (12) \\{{F_{bd}(t)} = {k_{bdf}{N_{bd}(t)}}} & (13) \\{\frac{\mathbb{d}{N_{ba}(t)}}{\mathbb{d}t} = {{{- k_{ba}}{N_{ba}(t)}} + {N_{{ba}\; 0}{u(t)}}}} & (14) \\{{F_{ba}(t)} = {k_{baf}{N_{ba}(t)}}} & (15)\end{matrix}$

The above formulas (4) to (15) are subjected to Laplace transform toderive a formula representing fluorescence within the donor wavelengthband (F_(donor)) and a formula representing fluorescence within theacceptor wavelength band (F_(acceptor)). The fluorescence F_(donor) andthe fluorescence F_(acceptor) are represented by the following formulas(16) and (17), respectively.

$\begin{matrix}{\mspace{79mu}\left\lbrack {{Formula}\mspace{14mu} 8} \right\rbrack} & \; \\{\mspace{79mu}{{F_{donor}(s)} = {\left( {\frac{k_{df}N_{d\; 0}\tau_{d}^{*}}{1 + {\tau_{d}^{*}s}} + \frac{k_{adf}N_{{ad}\; 0}\tau_{ad}}{1 + {\tau_{ad}s}} + \frac{k_{bdf}N_{{bd}\; 0}\tau_{bd}}{1 + {\tau_{bd}s}}} \right) \cdot {U(s)}}}} & (16) \\{{F_{acceptor}(s)} = {\left\{ {{k_{af}\left( {{\frac{k_{t}\tau_{a}}{1 + {\tau_{a}s}} \cdot \frac{N_{d\; 0}\tau_{d}^{*}}{1 + {\tau_{d}^{*}s}}} + \frac{N_{a\; 0}\tau_{a}}{1 + {\tau_{a}s}}} \right)} + \frac{k_{daf}N_{{da}\; 0}\tau_{da}}{1 + {\tau_{da}s}} + \frac{k_{baf}N_{{ba}\; 0}\tau_{ba}}{1 + {\tau_{ba}s}}} \right\} \cdot {U(s)}}} & (17)\end{matrix}$

wherein

τ_(d)*: fluorescence relaxation time constant of donor molecule at thetime when FRET occurs=1/(k_(d)+k_(t))

(k_(t)=0 (non-FRET state), τ_(d)*=τ_(d)=1/k_(d))

τ_(a): fluorescence relaxation time constant of acceptormolecule=1/k_(a)

τ_(ad): fluorescence relaxation time constant of acceptor moleculewithin donor wavelength band=1/k_(ad)

τ_(da): fluorescence relaxation time constant of donor molecule withinacceptor wavelength band=1/k_(da)

τ_(bd): fluorescence relaxation time constant of autofluorescence ofcell within donor wavelength band=1/k_(bd)

τ_(ba): fluorescence relaxation time constant of autofluorescence ofcell within acceptor wavelength band=1/k_(ba)

FIG. 8 is a diagram illustrating a model of dynamics of fluorescenceemission at the time when FRET occurs, which is represented by theformulas (16) and (17). It can be considered that the FRET sample 12emits fluorescence components indicated by routes W₁ to W₇ in FIG. 8. Inthe present invention, it can be considered that these fluorescencecomponents indicated by the routes W₁ to W₇ in the model shown in FIG. 8correspond to the fluorescence components (R₁ to R₇) introduced into thephotoelectric converters 27 a and 27 b shown in FIG. 4, respectively.

In the device 10, laser light output which is emitted from the laserlight source unit 22 is changed (modulated) into a sinusoidal wave athigh speed to irradiate the sample, and a fluorescence intensity signaloutput from the sample is processed at high speed to detect an amplitude(fluorescence intensity information) and a phase (phase information) percell. That is, frequency response characteristics of transfer functionsgiven by the formulas (16) and (17) are detected. When a fluorescencesignal is measured under the condition that a sinusoidal wave with anangular frequency ω_(M) is applied to the power u(t) of laser, thefluorescent signal is output as a sinusoidal wave having the sameangular frequency. The amplitude ratio and phase difference between aninput signal and an output signal can be expressed as the sum of vectorsin a complex plane when s in the formulas (16) and (17) is defined asjω_(M) and can be represented by the following formulas (18) and (19).

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 9} \right\rbrack & \; \\{\frac{F_{donor}(s)}{U(s)} = {{P_{1}{\mathbb{e}}^{{j\theta}_{1}}} + {P_{2}{\mathbb{e}}^{{j\theta}_{2}}} + {P_{3}{\mathbb{e}}^{{j\theta}_{3}}}}} & (18) \\{{{{\frac{F_{acceptor}(s)}{U(s)} = {{P_{7}{\mathbb{e}}^{{j\theta}_{7}}} + {P_{4}{\mathbb{e}}^{{j\theta}_{4}}} + {P_{5}{\mathbb{e}}^{{j\theta}_{5}}} + {P_{6}{\mathbb{e}}^{{j\theta}_{6}}}}}{{P_{1} = \frac{k_{df}N_{d\; 0}\tau_{d}^{*}}{\sqrt{1 + \left( {\tau_{d}^{*}\omega_{M}} \right)^{2}}}},\mspace{14mu}{\theta_{1} = {{- \tan^{- 1}}\tau_{d}^{*}\omega_{M}}}}{{P_{2} = \frac{k_{adf}N_{{ad}\; 0}\tau_{ad}}{\sqrt{1 + \left( {\tau_{ad}\omega_{M}} \right)^{2}}}},\mspace{14mu}{\theta_{2} = {{- \tan^{- 1}}\tau_{ad}\omega_{M}}}}{{P_{3} = \frac{k_{bdf}N_{{bd}\; 0}\tau_{bd}}{\sqrt{1 + \left( {\tau_{bd}\omega_{M}} \right)^{2}}}},\mspace{14mu}{\theta_{3} = {{- \tan^{- 1}}\tau_{bd}\omega_{M}}}}P_{7} = {k_{af}\frac{k_{t}\tau_{a}}{\sqrt{1 + \left( {\tau_{a}\omega_{M}} \right)^{2}}}\frac{N_{d\; 0}\tau_{d}^{*}}{\sqrt{1 + \left( {\tau_{d}^{*}\omega_{M}} \right)^{2}}}}},{\theta_{7} = {{- \left( {{\tan^{- 1}\tau_{a}\omega_{M}} + {\tan^{- 1}\tau_{d}^{*}\omega_{M}}} \right)} = {{- \tan^{- 1}}\frac{\left( {\tau_{a} + \tau_{d}^{*}} \right)\omega_{M}}{1 - {\tau_{a}\tau_{d}^{*}\omega_{M}^{2}}}}}}}{{P_{4} = \frac{k_{af}N_{a\; 0}\tau_{a}}{\sqrt{1 + \left( {\tau_{a}\omega_{M}} \right)^{2}}}},\mspace{14mu}{\theta_{4} = {{- \tan^{- 1}}\tau_{a}\omega_{M}}}}{{P_{5} = \frac{k_{daf}N_{{da}\; 0}\tau_{da}}{\sqrt{1 + \left( {\tau_{da}\omega_{M}} \right)^{2}}}},\mspace{14mu}{\theta_{5} = {{- \tan^{- 1}}\tau_{da}\omega_{M}}}}{{P_{6} = \frac{k_{baf}N_{{ba}\; 0}\tau_{ba}}{\sqrt{1 + \left( {\tau_{ba}\omega_{M}} \right)^{2}}}},\mspace{14mu}{\theta_{6} = {{- \tan^{- 1}}\tau_{ba}\omega_{M}}}}} & (19)\end{matrix}$

It goes without saying that the first term, second term, and third termof the formula (18) correspond to W₁, W₂, and W₃ in FIG. 8,respectively, and that the first term, second term, third term, andfourth term of the formula (19) correspond to W₇ (FRET component), W₄,W₅, and W₆ in FIG. 8, respectively.

It is difficult for conventional methods to quantitatively determine thefluorescence relaxation time constant τ_(d)* of a donor molecule at thetime when FRET occurs or the equivalent fluorescence relaxation timeconstant τ_(a)* (=tan θ₇/ω_(M)) of an acceptor molecule at the time whenFRET occurs. If τ_(d)* and τ_(a)* can be quantitatively determined, itis possible to detect the occurrence of FRET with a high degree ofaccuracy. As can be seen from the above relationships, τ_(d)* can bedetermined by determining P₁ and θ₁, and τ_(a)* can be determined bydetermining the above P₇ and θ₇.

Here, when the fluorescence intensity and phase of fluorescence withinthe donor wavelength band (including W₁ to W₃ shown in FIG. 8) aredefined as P_(donor) and θ_(donor), respectively, the formula (18) canbe transformed into the following formula (20). As can be seen from theformula (20), P₁ and θ₁ can be determined when the intensity P_(donor)and phase θ_(donor) of fluorescence within the donor wavelength band(including W₁ to W₃ shown in FIG. 8), P₂/P₄, P₄, P₃, and θ₃ are known.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 10} \right\rbrack & \; \\\begin{matrix}{{P_{1}{\mathbb{e}}^{{j\theta}_{1}}} = {{P_{donor}{\mathbb{e}}^{{j\theta}_{donor}}} - {P_{2}{\mathbb{e}}^{{j\theta}_{2}}} - {P_{3}{\mathbb{e}}^{{j\theta}_{3}}}}} \\{= {{P_{donor}{\mathbb{e}}^{{j\theta}_{donor}}} - {\frac{P_{2}}{P_{4}}P_{4}{\mathbb{e}}^{{j\theta}_{2}}} - {P_{3}{\mathbb{e}}^{{j\theta}_{3}}}}}\end{matrix} & (20)\end{matrix}$

Further, when the fluorescence intensity and phase of fluorescencewithin the acceptor wavelength band (including W₄ to W₇ shown in FIG. 8)are defined as P_(acceptor) and θ_(acceptor), respectively, the formula(19) can be transformed into the following formula (21). As can be seenfrom the formula (21), P₇ and θ₇ can be determined when the intensityP_(acceptor) and phase θ_(acceptor) of fluorescence within the acceptorwavelength band, P₅/P₁, P₁, P6, and θ₆ are known.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 11} \right\rbrack & \; \\\begin{matrix}{{P_{7}{\mathbb{e}}^{{j\theta}_{7}}} = {{P_{acceptor}{\mathbb{e}}^{{j\theta}_{acceptor}}} - {P_{4}{\mathbb{e}}^{{j\theta}_{4}}} - {P_{5}{\mathbb{e}}^{{j\theta}_{5}}} - {P_{6}{\mathbb{e}}^{{j\theta}_{6}}}}} \\{= {{P_{acceptor}{\mathbb{e}}^{{j\theta}_{acceptor}}} - {P_{4}{\mathbb{e}}^{{j\theta}_{4}}} - {\frac{P_{5}}{P_{1}}P_{1}{\mathbb{e}}^{{j\theta}_{5}}} - {P_{6}{\mathbb{e}}^{{j\theta}_{6}}}}}\end{matrix} & (21)\end{matrix}$

In the fluorescence relaxation time constant calculating unit 96 of theanalysis device 80, when the sample labeled with the donor molecule andthe acceptor molecule is irradiated with the above-mentioned laserlight, the fluorescence intensity P_(d) and phase θ_(d) of fluorescencewithin the donor wavelength band (including R₁ to R₃ shown in FIG. 5)and the fluorescence intensity P_(a) and phase θ_(a) of fluorescencewithin the acceptor wavelength band (including R₄ to R₇ shown in FIG. 5)are first calculated from the above-mentioned cos component and sincomponent. The measured values P_(d) and θ_(d) correspond to theabove-mentioned P_(donor) and θ_(donor), respectively, and P_(a) andθ_(a) likewise correspond to the above-mentioned P_(acceptor) andθ_(acceptor), respectively. The memory 84 of the analysis device 80stores values corresponding to the above-mentioned P₂/P₄, P₃ and θ₃, P₆and θ₆, and P₄ calculated by the first calibration unit 88 to the fourthcalibration unit 94 (which will be described later). Therefore, P₁ andθ₁ or P₇ and θ₇ are calculated using these values, and then τ_(d)*,τ_(a)*, and τ_(a), that is, the FRET fluorescence lifetime of donormolecule fluorescence component, the FRET fluorescence lifetime ofacceptor molecule fluorescence component, and the non-FRET fluorescencelifetime of acceptor molecule fluorescence component are determined fromthe definitional formulas of P_(i) and θ_(i) (i is an integer of 1 to 7)associated with the formulas (18) and (19) and the definitional formulaτ_(a)*=tan θ₇/ω_(M). At this time, the fluorescence relaxation timeconstant calculating unit 96 converts a formula expressed by phases andamplitudes, such as the formula (20) or (21), into a vector to performvector operation.

The FRET efficiency calculating unit 98 determines FRET efficiency E_(t)defined by the following formula (22) by using the fluorescencerelaxation time constant τ_(d) of a fluorescence component within thedonor wavelength band emitted by the donor molecule (corresponding tothe route R₁ shown in FIG. 4 in the non-FRET state). It is to be notedthat the fluorescence relaxation time constant τ_(d) is previouslydetermined by, for example, the second calibration unit 90 and thefourth calibration unit 94 (which will be described later) and stored inthe memory 84.[Formula 12]E _(t)=1−τ_(d)*/τ_(d)  (22)

The fluorescence relaxation time constant of the donor molecule isshorter when the degree of FRET is higher, that is, when energytransferred from the donor molecule to the acceptor molecule is larger.Such FRET efficiency E_(t) represents the degree of transfer of energyfrom the donor molecule to the acceptor molecule (the degree of FRET).

Further, in the present invention, FRET can be quantitatively measuredalso from measurement results within the acceptor wavelength band. Inthis case, as the fluorescence relaxation time constant τ_(a), a valuestored in the memory 84 can of course be used. The fluorescencerelaxation time constant τ_(a) is previously determined by, for example,the third calibration unit 92 and the fourth calibration unit 94 (whichwill be described later) and stored, and this value may be used.

By determining the fluorescence relaxation time constant τ_(d)* of thedonor molecule at the time when FRET occurs from the fluorescencerelaxation time constants τ_(a) and τ_(a)* of the acceptor molecule withthe use of the following formula (23), it is possible to multilaterallydetermine information on fluorescence relaxation time constant at thetime when FRET occurs not only from measurement results within the donorwavelength band but also from measurement results within the acceptorwavelength band.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 13} \right\rbrack & \; \\{{\tau_{a}^{*} \equiv {\tan\;{\theta_{t}/\omega_{M}}}} = \frac{\tau_{a} + \tau_{d}^{*}}{1 - {\tau_{a}\tau_{d}^{*}\omega_{M}^{2}}}} & (23)\end{matrix}$

Hereinbelow, each of the first calibration unit 88 to the fourthcalibration unit 94 will be described. When a sample, such as a cell,which has no fluorochrome attached thereto and which emitsautofluorescence is defined as an unlabeled sample, the firstcalibration unit 88 calculates fluorescence intensity information andphase information on autofluorescence emitted by the unlabeled sample.The first calibration unit 88 acquires detection values including phaseinformation from each of the photoelectric converters when the unlabeledsample as a measuring object is irradiated with laser light whoseintensity is modulated at a predetermined frequency, and calculatesfluorescence intensity information and phase information from thesedetection values.

More specifically, as shown in FIG. 9, the first calibration unit 88calculates, when the unlabeled sample (e.g., cell) is irradiated withlaser light, fluorescence intensity information and phase information onfluorescence within the donor wavelength band emitted by the cell itselfand on fluorescence within the acceptor wavelength band emitted by thecell itself. Here, it can be assumed that fluorescence emitted by thecell itself is not influenced by labels such as a donor molecule and anacceptor molecule. More specifically, a measured value P_(md1) offluorescence intensity of autofluorescence within the donor wavelengthband and a measured value θ_(md1) of phase of autofluorescence withinthe donor wavelength band obtained by the first calibration unit 88 areinformation on a fluorescence component indicated by R₃ in FIG. 5. Ameasured value P_(ma1) of fluorescence intensity of autofluorescencewithin the acceptor wavelength band and a measured value θ_(ma1) ofphase difference of autofluorescence within the acceptor wavelength bandare information on a fluorescence component indicated by R₆ in FIG. 5.These measured values can be directly used for the model of process ofFRET occurrence shown in FIG. 8, and relationships represented by thefollowing formulas (24) and (25) are established. The thus determined P₃and θ₃ and P₆ and θ₆ are stored in the memory 84.[Formula 14]P ₃ e ^(jθ) ³ =P _(md1) e ^(jθ) ^(md1)   (24)P ₆ e ^(jθ) ⁶ =P _(ma1) e ^(jθ) ^(ma1)   (25)

The second calibration unit 90 calculates the fluorescence intensity andphase of fluorescence emitted by a donor-labeled sample having only adonor fluorochrome. Further, the second calibration unit 90 calculatesthe ratio of the fluorescence intensity of a fluorescence componentwithin the acceptor wavelength band to the fluorescence intensity of afluorescence component within the donor wavelength band (leak rate ofdonor fluorescence) by using the calculated information on thefluorescence intensity and phase of the donor-labeled sample and thevalues of fluorescence intensity and phase of fluorescence componentsemitted by the unlabeled sample (P₃ and θ₃ and P₆ and θ₆) calculated bythe first calibration unit.

More specifically, as shown in FIG. 10, the second calibration unit 90calculates, when the donor-labeled sample is irradiated with laserlight, the fluorescence intensity and phase of fluorescence within thedonor wavelength band emitted by the donor molecule and the cell itselfand those of fluorescence within the acceptor wavelength, band emittedby the donor molecule and the cell itself. Here, when the measured valueof fluorescence intensity of the fluorescence within the donorwavelength band, the measured value of phase difference of thefluorescence within the donor wavelength band, the measured value offluorescence intensity of the fluorescence within the acceptorwavelength band, and the measured value of phase difference of thefluorescence within the acceptor wavelength band obtained by the secondcalibration unit 90 are defined as P_(md2), θ_(md2), P_(ma2), andθ_(ma2), respectively, the fluorescence intensity and phase offluorescence (indicated by R₁′ in FIG. 10) within the donor wavelengthband emitted by the donor molecule of the donor-labeled sample aredefined as P₁′ and θ₁′, respectively, and the fluorescence intensity andphase of fluorescence (indicated by R₅′ in FIG. 10) within the acceptorwavelength band emitted by the donor molecule of the donor-labeledsample are defined as P₅′ and θ₅′, respectively, relationshipsrepresented by the following formulas (26) and (27) are established. Asdescribed above, as the values of the fluorescence intensity and phaseof fluorescence components of autofluorescence, the above-mentionedvalues calculated by the first calibration unit can be directly used.[Formula 15]P ₁ ′e ^(jθ) ¹ ^(′) =P _(md2) e ^(jθ) ^(md2) −P ₃ e ^(jθ) ³   (26)P ₅ ′e ^(jθ) ⁵ ^(′) =P _(ma2) e ^(jθ) ^(ma2) −P ₆ e ^(jθ) ⁶   (27)

The second calibration unit 90 reads, from the memory 84, P₃ and θ₃ andP₆ and θ₆, which have been calculated by the first calibration unit 88,to calculate P₁′ and θ₁′ and P₅′ and θ₅′ by using the relationshipsrepresented by the formulas (26) and (27). The second calibration unit90 may also determine, from these results, the fluorescence relaxationtime constant τ_(d) of fluorescence within the donor wavelength bandemitted by the donor molecule at the time when FRET does not occur andthe fluorescence relaxation time constant τ_(da) of fluorescence withinthe acceptor wavelength band emitted by the donor molecule at the timewhen FRET does not occur.

As described above, when FRET occurs, radiative transition,non-radiative transition, and excitation energy transfer proceed at thesame time in the donor molecule. Further, when the amount of labelattached changes, fluorescence intensity also changes, and therefore P₁′and θ₁′ and P₅′ and θ₅′ cannot be regarded as information onfluorescence components indicated by R₁ and R₅ in FIG. 5 and thesemeasured values cannot be directly used for the model of process of FREToccurrence shown in FIG. 8. However, it can be assumed that the ratiobetween the fluorescence intensity of fluorescence within the donorwavelength band emitted by the donor molecule and the fluorescenceintensity of fluorescence within the acceptor wavelength band emitted bythe donor molecule, that is, P₅′/P₁′ (leak rate of donor fluorescence)is constant among different samples. More specifically, the ratio of P₅to P₁ in the formulas (20) and (21) can be represented as follows:P₅/P₁=P₅′/P₁′. The ratio P₅′/P₁′ (hereinafter, referred to as P₅/P₁)calculated by the second calibration unit 90 is stored in the memory 84.It is to be noted that at this time, P₁′ and θ₁′, P₅′ and θ₅′, thefluorescence relaxation time constant τ_(d) of fluorescence within thedonor wavelength band emitted by the donor molecule at the time whenFRET does not occur, and the fluorescence relaxation time constantτ_(da) of fluorescence within the acceptor wavelength band emitted bythe donor molecule at the time when FRET does not occur are also storedin the memory 84.

The third calibration unit 92 calculates the fluorescence intensity andphase of fluorescence emitted by a donor-labeled sample having only anacceptor fluorochrome. Further, the third calibration unit 92 calculatesthe ratio of the fluorescence intensity of a fluorescence componentwithin the donor wavelength band to the fluorescence intensity of afluorescence component within the acceptor wavelength band (leak rate ofacceptor fluorescence) by using the calculated information on thefluorescence intensity and phase of the acceptor-labeled sample and thevalues of fluorescence intensity and phase (P₃ and θ₃ and P₆ and θ₆) offluorescence components emitted by the unlabeled sample calculated bythe first calibration unit.

More specifically, as shown in FIG. 11, the third calibration unit 92calculates, when the acceptor-labeled sample is irradiated with laserlight, the fluorescence intensity and phase of fluorescence within thedonor wavelength band emitted by the acceptor molecule and the cellitself and those of fluorescence within the acceptor wavelength bandemitted by the acceptor molecule and the cell itself. When the measuredvalue of the fluorescence intensity of the fluorescence within the donorwavelength band, the measured value of phase difference of thefluorescence within the donor wavelength band, the measured value offluorescence intensity of the fluorescence within the acceptorwavelength band, and the measured value of phase difference of thefluorescence within the acceptor wavelength band obtained by the thirdcalibration unit 90 are defined as P_(md3), θ_(md3), P_(ma3), andθ_(ma3), respectively, the fluorescence intensity and phase offluorescence (indicated by R₂′ in FIG. 11) within the donor wavelengthband emitted by the acceptor molecule of the acceptor-labeled sample aredefined as P₂′ and θ₂′, respectively, and the fluorescence intensity andphase of fluorescence (indicated by R₄′ in FIG. 11) within the donorwavelength band emitted by the acceptor molecule of the acceptor-labeledsample are defined as P₄′ and θ₄′, respectively, relationshipsrepresented by the following formulas (28) and (29) are established. Asdescribed above, as the values of fluorescence intensity and phase offluorescence components of autofluorescence, the above-mentioned valuescalculated by the first calibration unit can be directly used.[Formula 16]P ₂ ′e ^(jθ) ² ^(′) =P _(md3) e ^(jθ) ^(md3) −P ₃ e ^(jθ) ³   (28)P ₄ ′e ^(jθ) ⁴ ^(′) =P _(ma3) e ^(jθ) ^(ma3) −P ₆ e ^(jθ) ⁶   (29)

The third calibration unit 92 reads, from the memory 84, P₃ and θ₃ andP₆ and θ₆, which have been calculated by the first calibration unit 88,to calculate P₂′ and θ₂′ and P₄′ and θ₄′ by using relationshipsrepresented by the following formulas (30) and (31). The thirdcalibration unit 92 may also determine, from these results, thefluorescence relaxation time constant τ_(a) of fluorescence within theacceptor wavelength band emitted by the acceptor molecule at the timewhen FRET does not occur and the fluorescence relaxation time constantτ_(ad) of fluorescence within the donor wavelength band emitted by theacceptor molecule at the time when FRET does not occur. As describedabove, when the amount of label attached changes, the fluorescenceintensity also changes, and therefore P₂′ and θ₂′ and P₄′ and θ₄′ cannotbe applied to the model for the occurrence of FRET shown in FIG. 8.However, it can be assumed that the ratio between the fluorescenceintensity of fluorescence within the acceptor wavelength band emitted bythe acceptor molecule and the fluorescence intensity of fluorescencewithin the donor wavelength band emitted by the acceptor molecule, thatis, P₂′/P₄′ (leak rate of acceptor fluorescence) is constant amongdifferent samples. More specifically, the ratio of P₂ to P₄ in theformulas (20) and (21) can be represented as follows: P₂/P₄=P₂′/P₄′. Theratio P₂′/P₄′ (hereinafter, referred to as P₂/P₄) calculated by thethird calibration unit 92 is stored in the memory 84. It is to be notedthat at this time, P₂′ and θ₂′, P₄′ and θ₄′, and the fluorescencerelaxation time constant τ_(a) of fluorescence within the acceptorwavelength band emitted by the acceptor molecule at the time when FRETdoes not occur and the fluorescence relaxation time constant τ_(ad) offluorescence within the donor wavelength band emitted by the acceptormolecule at the time when FRET does not occur are also stored in thememory 84.

The fourth calibration unit 94 calculates information on thefluorescence intensity and phase of fluorescence emitted by a non-FRETsample which has both a donor fluorescent molecule and an acceptorfluorescent molecule and which has been treated so as not to cause FRETby, for example, introducing a factor inactivating protein structuralchange or protein linkage. Further, the fourth calibration unit 94calculates the fluorescence intensity (represented by P₄″ in thefollowing formula (31)) and phase (represented by θ₄″ in the followingformula (31)) of fluorescence emitted by the acceptor-labeled sampledirectly excited by irradiation with laser light by using the calculatedinformation on the fluorescence intensity and phase of fluorescenceemitted by the non-FRET sample, the values of fluorescence intensity andphase of fluorescence components emitted by the unlabeled sample (P₃ andθ₃ and P₆ and θ₆) calculated by the first calibration unit, the leakrate of donor fluorescence (P₅/P₁) calculated by the second calibrationunit 90, and the leak rate of acceptor fluorescence (P₂/P₄) calculatedby the third calibration unit 92.

More specifically, as shown in FIG. 12, the fourth calibration unit 94calculates, when the non-FRET sample is irradiated with laser light, thefluorescence intensity and phase of fluorescence within the donorwavelength band emitted by the donor and acceptor molecules of thenon-FRET sample and the cell itself and the fluorescence intensity andphase of fluorescence within the acceptor wavelength band emitted by thedonor and acceptor molecules of the non-FRET sample and the cell itself,that is, P₁″, θ₁″, P₄″, and θ₄″ in the following formulas (30) and (31).When the measured value of fluorescence intensity of the fluorescencewithin the donor wavelength band, the measured value of phase differenceof the fluorescence within the donor wavelength band, the measured valueof fluorescence intensity of the fluorescence within the acceptorwavelength band, and the measured value of phase difference of thefluorescence within the acceptor wavelength band are defined as P_(md4),θ_(md4), P_(ma4), and θ_(ma4), respectively, the fluorescence intensityand phase of fluorescence (indicated by R₁″ in FIG. 12) within the donorwavelength band emitted by the donor molecule of the non-FRET sample aredefined as P₁″ and θ₁″, respectively, the fluorescence intensity andphase of fluorescence (indicated by R₂″ in FIG. 12) within the donorwavelength band emitted by the acceptor molecule of the non-FRET sampleare defined as P₂″ and θ₂″, respectively, the fluorescence intensity andphase of fluorescence (indicated by R₅″ in FIG. 12) within the acceptorwavelength band emitted by the donor molecule of the non-FRET sample aredefined as P₅″ and θ₅″, respectively, and the fluorescence intensity andphase of fluorescence (indicated by R₄″ in FIG. 12) within the acceptorwavelength band emitted by the acceptor molecule of the non-FRET sampleare defined as P₄″ and θ₄″, respectively, relationships represented bythe following formulas (30) and (31) are established. As describedabove, as the values of fluorescence intensity and phase of fluorescencecomponents of autofluorescence, the above-mentioned values calculated bythe first calibration unit can be directly used.

$\begin{matrix}\left\lbrack {{Formula}\mspace{14mu} 17} \right\rbrack & \; \\\begin{matrix}{{P_{1}^{''}{\mathbb{e}}^{{j\theta}_{1}^{''}}} = {{P_{{md}\; 4}{\mathbb{e}}^{{j\theta}_{{md}\; 4}}} - {P_{2}^{''}{\mathbb{e}}^{{j\theta}_{2}^{''}}} - {P_{3}{\mathbb{e}}^{{j\theta}_{3}}}}} \\{= {{P_{{md}\; 4}{\mathbb{e}}^{{j\theta}_{{md}\; 4}}} - {\frac{P_{2}^{''}}{P_{4}^{''}}P_{4}^{''}{\mathbb{e}}^{{j\theta}_{2}^{''}}} - {P_{3}{\mathbb{e}}^{{j\theta}_{3}}}}}\end{matrix} & (30) \\\begin{matrix}{{P_{4}^{''}{\mathbb{e}}^{{j\theta}_{4}^{''}}} = {{P_{{ma}\; 4}{\mathbb{e}}^{{j\theta}_{{ma}\; 4}}} - {P_{5}^{''}{\mathbb{e}}^{{j\theta}_{5}^{''}}} - {P_{6}{\mathbb{e}}^{{j\theta}_{6}}}}} \\{= {{P_{{ma}\; 4}{\mathbb{e}}^{{j\theta}_{{ma}\; 4}}} - {\frac{P_{5}^{''}}{P_{1}^{''}}P_{1}^{''}{\mathbb{e}}^{{j\theta}_{5}^{''}}} - {P_{6}{\mathbb{e}}^{{j\theta}_{6}}}}}\end{matrix} & (31)\end{matrix}$

As described above, as the ratio P₅″/P₁″ in the formula (31), the leakrate of donor fluorescence (P₅/P₁) calculated by the second calibrationunit 90 can be directly used (this is because the leak rate of donorfluorescence does not change depending on the amount of label attached).Likewise, as described above, as the ratio P₂″/P₄″ in the formula (30),the leak rate of acceptor fluorescence (P₂/P₄) calculated by the thirdcalibration unit 92 can be directly used. The fourth calibration unit 94reads, from the memory 84, P₃ and θ₃ and P₆ and θ₆ which have beencalculated by the first calibration unit 88, P₅/P₁ which has beencalculated by the second calibration unit 90, and P₂/P₄ which has beencalculated by the third calibration unit 92 to calculate P₄″ and θ₄″ byusing the relationships represented by the formulas (30) and (31). Atthe same time, P₁″ and θ₁″ are also calculated. As θ₂″, θ₂′ calculatedby the third calibration unit is used. As θ₅″, θ₅′ calculated by thesecond calibration unit is used. It is to be noted that the formulas(30) and (31) interact with each other through P₁″ and P₄″, but sincethe values of P₅″/P₁″ and P₂″/P₄″ are 0 to 1, solutions for P₄″ and θ₄″can be converged by, for example, iteration and are therefore easilydetermined.

P₄″ and θ₄″ are the fluorescence intensity and phase of fluorescenceemitted by the acceptor-labeled sample directly excited by irradiationwith laser light, which are calculated using a sample having a donormolecule and an acceptor molecule attached thereto. As can be seen alsofrom the formula (21), fluorescence emitted by the acceptor molecule atthe time when FRET occurs can be determined simply by adding the termrepresenting FRET fluorescence to the term representing fluorescenceemitted by the acceptor molecule by direct excitation. Therefore, themeasurement results (P₄″ and θ₄″) of the non-FRET sample cell (which hasboth an acceptor molecule and a donor molecule attached thereto) whoseamount of label is equivalent to that of a FRET sample to be measuredcan be directly used as the fluorescence intensity and phase offluorescence emitted by the acceptor-labeled sample directly excited byirradiation with laser light (i.e., P₄″=P₄ and θ₄″=θ₄).

As has been described above, the fluorescence relaxation time constantcalculating unit 96 determines τ_(d)* and τ_(a)* by using theinformation calculated by the first calibration unit 88 to the fourthcalibration unit 94 and stored in the memory 84 (e.g., the values ofP₂/P₄, P₃ and θ₃, P₆ and θ₆, and P₄), the measured values P_(donor) andθ_(donor) and P_(acceptor) and θ_(acceptor) of the FRET sample 12, andthe above formulas (20) and (21). Then, the FRET efficiency calculatingunit 98 determines FRET efficiency E_(t) represented by the formula(22).

Such a flow cytometer 10 as described above performs operations shown inFIG. 13 to determine FRET efficiency. The respective details of theseoperations are as described above. First, a FRET sample is prepared(Step S10). At this time, a plurality of FRET samples are suspended in ameasurement solution. The FRET sample solution forms a flow cell in thetube line 30 with a sheath liquid. The flow cell is irradiated withlaser light whose intensity is modulated at a predetermined frequency tomeasure fluorescence (Step S20).

The photoelectric converters 27 a and 27 b, which receive differentwavelength bands of fluorescence, start fluorescence measurement inresponse to a trigger signal generated by the light-receiving unit 24 toindicate the timing of the passage of the FRET sample 12 through ameasurement point in the tube line 30. A fluorescent signal obtained bythe measurement is processed in the phase difference detector 56 in thesignal processing unit 42 to extract a processing signal including thecos component of the fluorescence signal and a processing signalincluding the sin component of the fluorescence signal. These processingsignals are supplied to the controller 44, a high-frequency signal isremoved from each of the processing signals by the low-pass filter 62,and the cos component and the sin component of the fluorescence signalare determined by performing A/D conversion.

The thus determined cos component and sin component are supplied to theanalysis device 80 to calculate measurement information (fluorescenceintensity information and phase information on fluorescence within thedonor wavelength band emitted by each of the FRET samples, fluorescenceintensity information and phase information on fluorescence within theacceptor wavelength band of each of the FRET samples). Then, a scatterdiagram (two-dimensional correlation diagram) is displayed on thedisplay 94 (Step S30) by using the fluorescence intensity information onfluorescence within the donor wavelength band and the fluorescenceintensity information on fluorescence within the acceptor wavelengthband, each of information calculated within a predetermined measurementtime.

Then, in order to specify the samples in which FRET occurred, a samplegroup of the fluorescence region of the samples in which FRET occurredis selected in the scatter diagram displayed on the display 94. Theselection may be performed by using an input operation system such as amouse by an operator or may be performed automatically by, for example,the CPU 82. The representative values of fluorescence intensityinformation and phase information on the FRET samples included in theregion of the selected sample group (e.g., an average value, a gravitycenter value, and a frequency peak value) are determined (Step S40). Therepresentative values are used as the above-mentioned P_(d) and θ_(d)and P_(a) and θ_(a).

Then, the individual pieces of calibration information (the firstcalibration information to the fourth calibration information)previously calculated by the first calibration unit 88 to the fourthcalibration unit 94 and stored in the memory 84 are read from the memory84 of the analysis device 80 (Step S50).

Then, P₁ and θ₁ or P₇ and θ₇ are calculated by using the above-mentionedP_(d) and θ_(d) or P_(a) and θ_(a) and the individual pieces ofcalibration information to determine τ_(d)*, τ_(a)*, or τ_(a), that is,the FRET fluorescence lifetime of donor molecule fluorescence component,the FRET fluorescence lifetime of acceptor molecule fluorescencecomponent, or the non-FRET fluorescence lifetime of acceptor moleculefluorescence component. In the calculation, the fluorescence relaxationtime constant unit 96 converts a formula such as the formula (20) or(21) represented by phases and amplitudes to a vector to sequentiallyperform vector operation (Step S60).

Then, for example, FRET efficiency E_(t) is determined by using thefluorescence relaxation time constant τ_(d) of a fluorescence componentwithin the donor wavelength band emitted by the donor molecule (whichcorresponds to the route R₁ in FIG. 5 in non-FRET state) (Step S70).

FIG. 14 is a flowchart of first calibration carried out by the flowcytometer 10. In the first calibration, unlabeled samples are firstprepared (Step S110).

Then, the unlabeled samples are measured by the flow cytometer (StepS120). The measurement by the flow cytometer (Step S120), the display ofa scatter diagram (Step S130), and the selection of a sample group ofthe fluorescence region of the unlabeled samples (Step S140) are thesame operations as in Steps S20 to S40 shown in FIG. 13, and thereforedescriptions thereof are omitted here. In the first calibration,representative values extracted in Step S140 are defined as theabove-mentioned P_(md1) and θ_(md1) and P_(ma1) and θ_(ma1). Therepresentative values P_(md1) and θ_(md1) and P_(ma1) and θ_(ma1) ofmeasured information are stored as first calibration information in thememory 84 (Step S150). The first calibration information representsfluorescence intensity information P₃ and phase information θ₃ onfluorescence within the donor wavelength band emitted by the cell itselfwhen the unlabeled sample is irradiated with laser light andfluorescence intensity information P₆ and phase information θ₆ onfluorescence within the acceptor wavelength band emitted by the cellitself when the unlabeled sample is irradiated with laser light. It isto be noted that the details of the individual pieces of informationcalculated by the first calibration (first calibration information) andof the operations performed in the first calibration are as describedabove.

FIG. 15 is a flowchart of second calibration carried out by the flowcytometer 10. In the second calibration, donor-labeled samples are firstprepared (Step S210).

Then, the donor-labeled samples are measured by the flow cytometer (StepS210). The measurement by the flow cytometer (Step S220), the display ofa scatter diagram (Step S230), and the selection of a sample group ofthe fluorescence region of the donor-labeled samples (Step S240) are thesame operations as in Steps S20 to S40 shown in FIG. 13, and thereforedescriptions thereof are omitted here. In the second calibration,representative values extracted in Step S240 are defined as theabove-mentioned P_(md2) and θ_(md2) and P_(ma2) and θ_(ma2). Then, thefirst calibration information calculated by the first calibration unitis read from the memory 84 (Step S250). Then, second calibrationinformation is calculated by using P_(md2) and θ_(md2) and P_(ma2) andθ_(ma2) and the first calibration information (Step S260). The secondcalibration information includes P₅/P₁ representing the ratio of thefluorescence intensity of a fluorescence component within the acceptorfluorescence wavelength band emitted by the donor-labeled sample to thefluorescence intensity of a fluorescence component within the donorwavelength band emitted by the donor-labeled sample (i.e., leak rate ofdonor fluorescence). At this time, as described above, the fluorescencerelaxation time constant τ_(d) of fluorescence within the donorwavelength band emitted by the donor molecule at the time when FRET doesnot occur and the fluorescence relaxation time constant τ_(da) offluorescence within the acceptor wavelength band emitted by the donormolecule at the time when FRET does not occur are also determined. Theseconstants are also included in the second calibration information. Thesecond calibration information is stored in the memory 84 (Step S270).It is to be noted that the details of the second calibration informationcalculated by the second calibration and of the operations performed inthe second calibration are as described above.

FIG. 16 is a flowchart of third calibration carried out by the flowcytometer 10. In the third calibration, acceptor-labeled samples arefirst prepared (Step S310).

Then, the acceptor-labeled samples are measured by the flow cytometer(Step S320). The measurement by the flow cytometer (Step S320), thedisplay of a scatter diagram (Step S330), and the selection of a samplegroup of the fluorescence region of the acceptor-labeled samples (StepS340) are the same operations as in Steps S20 to S40 shown in FIG. 13,and therefore descriptions thereof are omitted here. In the thirdcalibration, representative values extracted in Step S340 are defined asthe above-mentioned P_(md3) and θ_(md3) and P_(ma3) and θ_(ma3). Then,the first calibration information calculated by the first calibrationunit is read from the memory 84 (Step S350). Then, third calibrationinformation is calculated by using the P_(md3) and θ_(md3) and P_(ma3)and θ_(ma3) and the first calibration information (Step S360). Thesecond calibration information includes P₂/P₄ representing the ratio ofthe fluorescence intensity of a fluorescence component within the donorfluorescence wavelength band emitted by the acceptor-labeled sample tothe fluorescence intensity of a fluorescence component within theacceptor wavelength band emitted by the acceptor-labeled sample (i.e.,leak rate of acceptor fluorescence). At this time, as described above,the fluorescence relaxation time constant τ_(a) of fluorescence withinthe acceptor wavelength band emitted by the acceptor molecule at thetime when FRET does not occur and the fluorescence relaxation timeconstant τ_(ad) of fluorescence within the donor wavelength band emittedby the acceptor molecule at the time when FRET does not occur are alsodetermined. These constants are also included in the third calibrationinformation. The third calibration information is stored in the memory84 (Step S370). It is to be noted that the details of the thirdcalibration information calculated by the third calibration and of theoperations performed in the third calibration are as described above.

FIG. 17 is a flowchart of fourth calibration carried out by the flowcytometer 10. In the fourth calibration, non-FRET samples are firstprepared (Step S410).

Then, the non-FRET samples are measured by the flow cytometer (StepS420). The measurement by the flow cytometer (Step. S420), the displayof a scatter diagram (Step S430), and the selection of a sample group ofthe fluorescence region of the non-FRET samples (Step S440) are the sameoperations as in Steps S20 to S40 shown in FIG. 13, and thereforedescriptions thereof are omitted here. In the fourth calibration,representative values extracted in Step S440 are defined as theabove-mentioned P_(md4) and θ_(md4) and P_(ma4) and θ_(ma4). Then, thefirst to third calibration information calculated by the first to thirdcalibration units is read from the memory 84 (Step S450). Then, fourthcalibration information is calculated by using P_(md4) and θ_(md4) andP_(ma4) and θ_(ma4) and the first to third calibration information (StepS460). The fourth calibration information includes information on thefluorescence intensity P₄ and phase θ₄ of fluorescence emitted by theacceptor-labeled sample directly excited by irradiation with laserlight. The thus calculated fourth calibration information is stored, inthe memory 84 (Step S470). The individual pieces of calibrationinformation calculated by the first to fourth calibrations are used forFRET detection performed using FRET samples according to the flowchartshown in FIG. 13. It is to be noted that the details of the individualpieces of information determined by the fourth calibration (fourthcalibration information) and of the operations performed in the fourthcalibration are as described above.

As described above, in the present invention, the leak rate of donormolecule fluorescence (P₅/P₁) and the leak rate of acceptor fluorescence(P₂/P₄), which do not change depending on the amount of label attachedto a target of FRET measurement, are previously calculated using samples(e.g., donor-labeled samples and acceptor-labeled samples) differentfrom the FRET samples in the amount of label attached. By processingmeasured information on the FRET samples using suchpreviously-calculated information such as the leak rates, it is possibleto determine the fluorescence lifetime of the FRET sample with a highdegree of accuracy. Further, fluorescence components emitted by theacceptor molecule directly excited by irradiation with laser light arepreviously determined using non-FRET samples treated so as not to causeFRET. By processing measured information on the FRET samples using suchpreviously-calculated information, it is possible to determine thefluorescence lifetime of the FRET sample with a high degree of accuracy.Further, by removing autofluorescence components emitted by the cellitself having the donor molecule and the acceptor molecule attachedthereto, it is possible to determine the fluorescence lifetime of theFRET sample with a higher degree of accuracy.

It is to be noted that the above embodiment uses non-FRET samples, eachof which has both the donor fluorescent molecule and the acceptorfluorescent molecule and has been treated so as not to cause FRET byintroducing a factor which inactivates protein structural change orprotein linkage. By calculating information on the fluorescenceintensity and phase of fluorescence emitted by the non-FRET sample, thefluorescence intensity and phase of fluorescence components emitted bythe acceptor-labeled sample directly excited by irradiation with laserlight are determined. For example, in a case where a sample labeled withthe acceptor molecule is irradiated with two laser beams with differentwavelengths so that fluorescence is emitted by the acceptor moleculeexcited by irradiation with the laser beams, it can be assumed that theratio between fluorescence components within one wavelength band emittedby irradiation with the two laser beams is constant irrespective of theamount of label attached to a sample to be measured. More specifically,it can be assumed that when the ratio between fluorescence componentsemitted by the FRET sample by irradiation with the two laser beams ischanged relative to the ratio between fluorescence components emitted bythe acceptor-labeled sample by irradiation with the two laser beams, theamount of change is due to FRET. It can be said, when the ratio betweenfluorescence components emitted by the acceptor-labeled sample byirradiation with the two laser beams is previously calculated,fluorescence intensity information and phase information on fluorescencecomponents emitted by the acceptor-labeled sample directly excited bylaser can be determined by calculating the ratio between fluorescencecomponents emitted by the FRET sample by irradiation with the two laserbeams. In the present invention, fluorescence intensity information andphase information on fluorescence components emitted by theacceptor-labeled sample directly excited by laser may be calculated bysuch a method. It is to be noted that as a means for detectingfluorescence components emitted by irradiation with two laser beamsindependently, a means described in Japanese Patent Application Nos.2005-37399 and 2006-054347, which are previous patent applications bythe present inventors, can be used.

The FRET detection method and FRET detection device according to thepresent invention have been described in detail, but the presentinvention is not limited to the above-described embodiments and, variouschanges and modifications can be made without departing from the scopeand sprit of the present invention.

What is claimed is:
 1. A FRET detection method of detecting FRET (Fluorescence Resonance Energy Transfer) in which energy of a donor molecule is transferred to an acceptor molecule, the method comprising the steps of: a) measuring fluorescence emitted from each of samples by two or more detection sensors having different light-receiving wavelength bands, each of the samples being labeled with a donor molecule and an acceptor molecule and being irradiated with laser light whose intensity is modulated at a predetermined frequency, to acquire detection values including fluorescence intensity information and phase information on the fluorescence emitted from each of the samples; b) reading calibration information previously stored in a memory means, which includes at least a first intensity ratio that is a ratio between fluorescence intensities at the light-receiving wavelength bands of a donor molecule fluorescence component emitted from the donor molecule included in the fluorescence emitted from each of the samples, phase information on the donor molecule fluorescence component relative to the modulated laser light, a second intensity ratio that is a ratio between fluorescence intensities at the light-receiving wavelength bands of an acceptor molecule fluorescence component emitted from the acceptor molecule included in the fluorescence, phase information on the acceptor molecule fluorescence component relative to the laser light, and a non-FRET fluorescence lifetime of the donor molecule fluorescence component when the FRET does not occur, which is a lifetime defined by assuming that fluorescence emitted from the donor molecule excited by laser light is a relaxation response of a first-order lag system; c) calculating fluorescence intensity information and phase information on the fluorescence at each of the light-receiving wavelength bands, based on the detection values, and determining a FRET fluorescence lifetime of the donor molecule fluorescence component, which is defined by assuming that fluorescence emitted from the donor molecule excited by laser light is a relaxation response of a first-order lag system, using the calculated fluorescence intensity information, the calculated phase information, the first intensity ratio, the phase information on the donor molecule fluorescence component, the second intensity ratio, the phase information on the acceptor molecule fluorescence component; and d) determining information on FRET occurrence using a ratio between the FRET fluorescence lifetime of the donor molecule fluorescence component and the non-FRET fluorescence lifetime of the donor molecule fluorescence component.
 2. The FRET detection method according to claim 1, wherein in the step d), FRET efficiency E_(t) is determined as the information on the occurrence of FRET, which is represented by 1−(τ_(d)*/τ_(d)), wherein τ_(d) is the non-FRET fluorescence lifetime of the donor molecule fluorescence component and τ_(d)* is the FRET fluorescence lifetime of the donor molecule fluorescence component.
 3. The FRET detection method according to claim 1, wherein in the step c), fluorescence intensity information and phase information on each of the samples are calculated based on each of the detection values and the FRET fluorescence lifetime is determined based on the calculated multiple pieces of fluorescence intensity information and phase information.
 4. The FRET detection method according to claim 1, wherein in the step c), the fluorescence intensity information and the phase information calculated at each of the light-receiving wavelength bands are represented as a vector, and fluorescence intensity information and phase information on the donor molecule fluorescence component and fluorescence intensity information and phase information on a FRET component of an acceptor molecule fluorescence component which is emitted by the acceptor molecule on the occurrence of FRET are calculated at, and the FRET fluorescence lifetime is determined by using the calculated information.
 5. The FRET detection method according to claim 4, further comprising the steps of: preparing a predetermined sample which is each of the samples unlabeled with the donor molecule and the acceptor molecule and emits autofluorescence when irradiated with the laser light; measuring, at each of the light-receiving wavelength bands, the autofluorescence emitted by the predetermined sample which is irradiated with the laser light; calculating fluorescence intensity information and phase information on the autofluorescence from the measured autofluorescence within each of the light-receiving wavelength bands to store the calculated fluorescence intensity information and phase information in the memory means; wherein in the step b), the stored fluorescence intensity information and the phase information on the autofluorescence are read from the memory means and the read fluorescence intensity information and the phase information on the autofluorescence are represented as a vector, and in the step c), the vector of the autofluorescence is subtracted from each vector at the light-receiving wavelength bands of the samples to be measured, and the FRET fluorescence lifetime is determined using a vector obtained by the subtraction.
 6. The FRET detection method according to claim 5, wherein the autofluorescence is measured by each of the detection sensors by irradiating the predetermined sample as a measuring object with laser light whose intensity is modulated at a predetermined frequency.
 7. The FRET detection method according to claim 4, wherein the light-receiving wavelength bands include a first wavelength band centered around a peak wavelength at which a fluorescence intensity of the donor molecule fluorescence component is maximum and a second wavelength band centered around a peak wavelength at which a fluorescence intensity of the acceptor molecule fluorescence component is maximum, in the step c), fluorescence intensity information and phase information on the donor molecule fluorescence component emitted are calculated using at least the second intensity ratio and the vector at the first wavelength band represented by the detection values acquired from the detection sensors with the first wavelength band, and fluorescence intensity information and phase information on the FRET component are calculated using at least the first intensity ratio and the vector at the second wavelength band represented by the detection values acquired from the detection sensors with the second wavelength band.
 8. The FRET detection method according to claim 7, wherein the memory means previously stores fluorescence intensity information and phase information on a directly-excited fluorescence component of the acceptor molecule fluorescence component, the directly-excited fluorescence component being emitted by the acceptor molecule directly excited by the laser light, in the step b), fluorescence intensity information and phase information on the directly-excited fluorescence component are read from the memory means to represent the information on the directly-excited fluorescence component as a vector, and in the step c), fluorescence intensity information and phase information on the acceptor molecule fluorescence component emitted at the time when the FRET occurs are calculated using at least the vector of the directly-excited fluorescence component and the vector at the second wavelength band.
 9. The FRET detection method according to claim 7, wherein the memory means previously stores fluorescence intensity information and phase information on a directly-excited fluorescence component of the acceptor molecule fluorescence component, the directly-excited fluorescence component being emitted by the acceptor molecule directly excited by the laser light, in the step b), fluorescence intensity information and phase information on the directly-excited fluorescence component are read from the memory means and then represented as a vector of the directly-excited fluorescence component, and in the step c), phase information on the FRET component is further calculated using at least the vector at the second wavelength band and the first intensity ratio, and a FRET fluorescence lifetime of the acceptor molecule fluorescence component emitted at the time when the FRET occurs and a non-FRET fluorescence lifetime of the acceptor molecule fluorescence component emitted at the time when the FRET does not occur are determined using the calculated phase information on the FRET component and the vector of the directly-excited fluorescence component, and a FRET fluorescence lifetime of the donor molecule fluorescence component is determined using the FRET fluorescence lifetime of the acceptor molecule fluorescence component and the non-FRET fluorescence lifetime of the acceptor molecule fluorescence component.
 10. The FRET detection method according to claim 1, further comprising the steps of: preparing a non-FRET sample which is labeled with the donor molecule and the acceptor molecule and which has been treated not to cause FRET; measuring, at each of the light-receiving wavelength bands, fluorescence emitted by the non-FRET sample which is irradiated with the laser light; and calculating fluorescence intensity information and phase information on the a directly-excited fluorescence component emitted by the acceptor molecule directly excited by laser light within each of the light-receiving wavelength bands from the measured fluorescence, using the first intensity ratio previously stored in the memory means, the phase information on the donor molecule fluorescence component, the second intensity ratio previously stored in the memory means, and the phase information on the acceptor molecule fluorescence component, to store the calculated fluorescence intensity information and phase information in the memory means.
 11. The FRET detection method according to claim 10, wherein the non-FRET sample is a sample obtained by labeling, with the donor molecule and the acceptor molecule, a predetermined sample which emits autofluorescence when excited by the laser light, the method further comprising the steps of: preparing the predetermined sample; measuring, at each of the light-receiving wavelength bands, the autofluorescence emitted by the predetermined sample which is irradiated with the laser light; calculating fluorescence intensity information and phase information on the autofluorescence from the measured autofluorescence within each of the light-receiving wavelength bands, to store the calculated fluorescence intensity information and phase information on the autofluorescence in the memory means; subtracting an autofluorescence vector representing the fluorescence intensity information and phase information on the autofluorescence from a non-FRET sample vector representing fluorescence intensity information and phase information on fluorescence emitted by the non-FRET sample; and calculating a directly-excited fluorescence component vector representing the fluorescence intensity information and phase information on the directly-excited fluorescence component, using a vector obtained by the subtraction.
 12. The FRET detection method according to claim 1, the method further comprising the steps of: preparing a donor molecule sample which is labeled with only the donor molecule; measuring, at each of the light-receiving wavelength bands, fluorescence emitted by the donor molecule of the donor molecule sample which is irradiated with the laser light; calculating fluorescence intensity information and phase information on the measured fluorescence emitted by the donor molecule sample within each of the light-receiving wavelength bands, to obtain the first intensity ratio; and storing the phase information on the measured fluorescence and the first intensity ratio.
 13. The FRET detection method according to claim 12, wherein the donor molecule sample is a sample obtained by labeling, with the donor molecule, a predetermined sample which emits autofluorescence when excited by the laser light, the method further comprising the steps of: preparing the predetermined sample; measuring, at each of the light-receiving wavelength bands, the autofluorescence emitted by the predetermined sample which is irradiated with the laser light; calculating fluorescence intensity information and phase information on the autofluorescence from the measured autofluorescence within each of the light-receiving wavelength bands to store the calculated fluorescence intensity information and phase information in the memory means; subtracting an autofluorescence vector representing the fluorescence intensity information and phase information on the autofluorescence from a donor molecule sample vector representing fluorescence intensity information and phase information on fluorescence emitted by the donor molecule sample; and calculating the fluorescence intensity information and phase information on the fluorescence emitted by the donor molecule sample, using a vector obtained by the subtraction.
 14. The FRET detection method according to claim 1, the method further comprising the steps of: preparing an acceptor molecule sample which is labeled with only the acceptor molecule; measuring, at each of the light-receiving wavelength bands, fluorescence emitted by the acceptor molecule of the acceptor molecule sample which is irradiated with the laser light; calculating fluorescence intensity information and phase information on the measured fluorescence emitted by the acceptor molecule sample within each of the light-receiving wavelength bands, to obtain the second intensity ratio; and storing the phase information on the measured fluorescence and the second intensity ratio.
 15. The FRET detection method according to claim 14, wherein the acceptor molecule sample is a sample obtained by labeling, with the acceptor molecule, a predetermined sample which emits autofluorescence when excited by the laser light, the method further comprising the steps of: preparing the predetermined sample; measuring, at each of the light-receiving wavelength bands, the autofluorescence emitted by the predetermined sample which is irradiated with the laser light; calculating fluorescence intensity information and phase information on the autofluorescence from the measured autofluorescence within each of the light-receiving wavelength bands, to store the calculated fluorescence intensity information and phase information in the memory means; subtracting an autofluorescence vector representing the fluorescence intensity information and phase information on the autofluorescence from an acceptor molecule sample vector representing fluorescence intensity information and phase information on fluorescence emitted by the acceptor molecule sample; and calculating the fluorescence intensity information and phase information on the fluorescence emitted by the acceptor molecule sample, using a vector obtained by the subtraction.
 16. A device for detecting FRET (Fluorescence Resonance Energy Transfer), in which energy of a donor molecule transfers to an acceptor molecule, the device comprising: an information acquiring unit which acquires detection values, each values including fluorescence intensity information and phase information on fluorescence emitted by each of the samples to be measured by allowing two or more detection sensors different in light-receiving wavelength band to receive fluorescence emitted by each of the samples to be measured, each of the samples being labeled with a donor molecule and an acceptor molecule and being irradiated with laser light whose intensity is modulated at a predetermined frequency; a memory means for previously storing calibration information including at least a first intensity ratio that is a ratio between fluorescence intensities at the light-receiving wavelength bands of a donor molecule fluorescence component emitted by the donor molecule included in the fluorescence emitted by each of the samples to be measured, phase information on the donor molecule fluorescence component, an acceptor intensity ratio that is a ratio between fluorescence intensities at the light-receiving wavelength bands of an acceptor molecule fluorescence component emitted by the acceptor molecule, phase information of the acceptor molecule fluorescence component, and a non-FRET fluorescence lifetime of the donor molecule fluorescence component emitted at the time when the FRET does not occur, which is a lifetime defined by assuming that fluorescence emitted by the donor molecule excited by laser light is a relaxation response of a first-order lag system; a FRET fluorescence lifetime calculating unit which calculates fluorescence intensity information and phase information on fluorescence within each of the light-receiving wavelength bands emitted by each of the samples to be measured based on the detection values acquired by the information acquiring unit, and determines a FRET fluorescence lifetime of the donor molecule fluorescence component, which is defined by assuming that fluorescence emitted by the donor molecule excited by laser light is a relaxation response of a first-order lag system, by using the calculated fluorescence intensity information, the calculated phase information, the first intensity ratio read from the memory means, the phase information on the donor molecule fluorescence component, the second intensity ratio, and the phase information on the acceptor molecule fluorescence component; and a FRET occurrence information calculating unit which determines information on the occurrence of FRET represented by a ratio between the FRET fluorescence lifetime of the donor molecule fluorescence component and the non-FRET fluorescence lifetime of the donor molecule fluorescence component.
 17. The FRET detection device according to claim 16, wherein in the FRET fluorescence lifetime calculating unit, the fluorescence intensity information and the phase information calculated from each of the detection values are represented as a vector, and fluorescence intensity information and phase information on the donor molecule fluorescence component and fluorescence intensity information and phase information on a FRET component of an acceptor molecule fluorescence component which is emitted by the acceptor molecule at the time when the FRET occurs, are calculated using the vector, the first intensity ratio, the phase information on the donor molecule fluorescence component, the second intensity ratio of the acceptor molecule fluorescence component, and the phase information on the acceptor molecule fluorescence component, and the FRET fluorescence lifetime is determined by using the calculated information.
 18. The FRET detection device according to claim 16, wherein the respective light-receiving wavelength bands of the sensors are a first wavelength band centered around a peak wavelength at which a fluorescence intensity of the donor molecule fluorescence component is maximum and a second wavelength band centered around a peak wavelength at which a fluorescence intensity of the acceptor molecule fluorescence component is maximum, and the FRET fluorescence lifetime calculating unit calculates fluorescence intensity information and phase information on the donor molecule fluorescence component by using at least a vector at the first wavelength band determined from the detection value acquired from one of the detection sensors with the first wavelength band and the second intensity ratio, and calculates fluorescence intensity information and phase information on the FRET component by using at least a vector at the second wavelength band represented by the detection value acquired from one of the detection sensors with the second wavelength band and the first intensity ratio.
 19. The FRET detection device according to claim 16, further comprising an autofluorescence calibration unit, wherein the autofluorescence calibration unit acquires a detection value including fluorescence intensity information and phase information at each of the light-receiving wavelength bands from each of the detection sensors by irradiating, with the laser, light a predetermined sample which is each of the samples unlabeled with the donor molecule and the acceptor molecule and emits autofluorescence when irradiated with the laser light, calculates fluorescence intensity information and phase information on the autofluorescence within each of the light-receiving wavelength bands, and stores the calculated fluorescence intensity information and the calculated phase information on the autofluorescence in the memory means, and in the FRET fluorescence lifetime calculating unit, a vector representing the fluorescence intensity information and the phase information on the autofluorescence is subtracted from a vector representing information on fluorescence within each of the light-receiving wavelength bands emitted by the sample to be measured, and the FRET fluorescence lifetime is determined using a vector obtained by the subtraction.
 20. The FRET detection device according to claim 16, further comprising a non-FRET calibration unit, wherein the non-FRET calibration unit calculates fluorescence intensity information and phase information on fluorescence within each of the light-receiving wavelength bands emitted by a non-FRET sample, which has the donor and acceptor molecules attached thereto and which has been treated so as not to cause FRET, when a detection value including fluorescence intensity information and phase information at each of the light-receiving wavelength bands is acquired from each of the detection sensors by irradiating the non-FRET sample with the laser light, and calculates fluorescence intensity information and phase information on a directly-excited fluorescence component emitted by the acceptor molecule directly excited by the laser light by using the calculated fluorescence intensity information, the calculated phase information, the first intensity ratio, the phase information on the donor molecule fluorescence component, the second intensity ratio, and the phase information on the acceptor molecule fluorescence component, and derives a directly-excited fluorescence component vector representing the calculated information, and stores a derived result in the memory means, and the FRET fluorescence lifetime calculating unit determines the FRET fluorescence lifetime by using the directly-excited fluorescence component vector.
 21. The FRET detection device according to claim 16, further comprising a donor molecule calibration unit, wherein the donor molecule calibration unit calculates fluorescence intensity information and phase information on fluorescence within each of the light-receiving wavelength bands emitted by a donor molecule sample, which is each of the samples labeled with only the donor molecule, when the donor molecule sample irradiated with the laser light emits fluorescence and a detection value including fluorescence intensity information and phase information on the fluorescence within each of the light-receiving wavelength bands emitted by the donor molecule sample is acquired from each of the detection sensors, and calculates the first intensity ratio, and stores the calculated first intensity ratio and the calculated phase information on fluorescence emitted by the donor molecule sample in the memory means.
 22. The FRET detection device according to claim 16, further comprising a second molecule calibration unit, wherein the second molecule calibration unit calculates fluorescence intensity information and phase information on fluorescence within each of the light-receiving wavelength bands emitted by an acceptor molecule sample, which is each of the samples labeled with only the acceptor molecule, when the acceptor molecule sample irradiated with the laser light emits fluorescence and a detection value including fluorescence intensity information and phase information on the fluorescence within each of the light-receiving wavelength bands emitted by the acceptor molecule sample is acquired from each of the detection sensors, and calculates, the second intensity ratio, and stores the calculated second intensity ratio and the calculated phase information on fluorescence emitted by the acceptor molecule sample in the memory means. 